RNAi agents and compositions for inhibiting expression of apolipoprotein C-III (APOC3)

ABSTRACT

The present disclosure relates to RNAi agents, e.g., double stranded RNAi agents, capable of inhibiting Apolipoprotein C-III (also called APOC3, apoC-III, APOC-III, and APO C-III) gene expression, and compositions that include APOC3 RNAi agents. The APOC3 RNAi agents disclosed herein may be conjugated to targeting ligands, including ligands that include N-acetyl-galactosamine, to facilitate the delivery to cells, including to hepatocytes. Pharmaceutical compositions that include one or more APOC3 RNAi agents, optionally with one or more additional therapeutics, are also described. Delivery of the APOC3 RNAi agents in vivo provides for inhibition of APOC3 gene expression, and can result in lower triglycerides and/or cholesterol levels in the subject. The APOC3 RNAi agents can be used in methods of treatment of APOC3-related diseases and disorders, including hypertriglyceridemia, cardiovascular disease, and other metabolic-related disorders and diseases.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional PatentApplication Ser. No. 62/720,434, filed on Aug. 21, 2018, U.S.Provisional Patent Application Ser. No. 62/643,927, filed on Mar. 16,2018, and U.S. Provisional Patent Application Ser. No. 62/556,818, filedon Sep. 11, 2017, the contents of each of which are incorporated hereinby reference in their entirety.

SEQUENCE LISTING

This application contains a Sequence Listing which has been submitted inASCII format and is hereby incorporated by reference in its entirety.The ASCII copy is named 30655_SeqListUS1 and is 195 kb in size.

FIELD OF THE INVENTION

The present disclosure relates to RNA interference (RNAi) agents, e.g.,double stranded RNAi agents, for inhibition of apolipoprotein C-Ill geneexpression, compositions that include apolipoprotein C-III RNAi agents,and methods of use thereof.

BACKGROUND

Apolipoprotein C-III (also called APOC3, apoC-III, APOC-III, and APOC-III), encoded by the human Apolipoprotein C-III gene, has recentlyemerged as a promising target for the treatment of diseases associatedwith hypertriglyceridemia. Elevated serum triglyceride (TG) levels havebeen identified as an independent risk factor for cardiovasculardisease, and as a contributing factor in the development ofatherosclerosis. Individuals with severe hypertriglyceridemia(often >1000 mg/dL) are also at risk of recurrent pancreatitis.Triglycerides are primarily transported in the blood as a majorcomponent of very low density lipoprotein (VLDL) and chylomicronparticles, which are known as TG-rich lipoproteins. Lipoproteins arecomposed of a hydrophobic triacylglycerol and cholesteryl ester core,and a hydrophilic outer layer of phospholipids, cholesterol, andapoproteins. APOC3 is one of these apoproteins.

APOC3 is primarily synthesized in the liver and plays an important rolein the production, metabolism, and clearance of TG-rich lipoproteinsfrom plasma. Several gain-of-function polymorphisms have been identifiedin the promoter region of the APOC3 gene, which are postulated to becontributing factors in development of hypertriglyceridemia (See, e.g.,Wang, Y., et al., Association of Apolipoprotein C3 Genetic Polymorphismswith the Risk of Ischemic Stroke in the Northern Chinese Han Population,11 PLoS One e0163910 (2016); Li, Y., et al., Apolipoprotein C3 genevariants and the risk of coronary heart disease: A meta-analysis 9 MetaGene 104-109 (2016)). Increased APOC3 synthesis in the liver promotessecretion of TG-rich VLDL. In addition, over-abundance of APOC3 inhibitsthe activity of lipoprotein lipase and hepatic lipase, furtherincreasing serum TG levels by delaying the catabolism of TG-richlipoproteins. Furthermore, elevated APOC3 also delays the hepaticclearance of TG-rich lipoprotein and their remnant particles byinterfering with their binding to hepatic receptors. Several largegenetic analysis studies have reported that individuals withloss-of-function mutations of APOC3 exhibit low levels of triglycerideand reduced incidence of cardiovascular disease. (See, e.g., BernelotMoens, S. J., et al., Inhibition of ApoCIII: the next PCSK9? 25 CurrOpin Lipidol 418-422 (2014); Saleheen, D., et al., Human knockouts andphenotypic analysis in a cohort with a high rate of consanguinity, 544Nature 235-239 (2017)).

Currently, hypertriglyceridemia is often treated with fibrates or incombination with statins in moderate cases; however, in most cases, thereduction in serum TG is modest. Additionally, available therapeuticsare often ineffective in patients with monogenic causes of very severehypertriglyceridemia (such as patients with familial chylomicronemiasyndrome) because the disease-causing mutations lead to dysfunctionallipoprotein lipase and functional lipoprotein lipase is required foroptimal response to standard therapies. There is a need for an effectivetherapeutic that can provide a substantial TG lowering effect for thetreatment of diseases where APOC3 may play a role, such ashypertriglyceridemia induced pancreatitis, metabolic syndrome, type IIdiabetes mellitus, familial chylomicronemia syndrome, familial partiallipodystrophy, obesity, hyperlipidemia, hypertriglyceridemia, abnormallipid and/or cholesterol metabolism, atherosclerosis, cardiovasculardisease, coronary artery disease, and other metabolic-related disordersand diseases. Certain other APOC3-specific RNA interference (RNAi)agents have been shown to inhibit expression of APOC3 gene expression,for example, in International Patent Application Publication No. WO2016/011123 A1, to Weiler et al., which is incorporated herein byreference in its entirety. The APOC3 RNAi agents disclosed herein,however, were not previously disclosed or known and provide for highlypotent and efficient inhibition of the expression of an APOC3 gene.

SUMMARY

There exists a need for novel APOC3 RNA interference (RNAi) agents (alsoherein termed RNAi agent, RNAi trigger, or trigger) that are able toselectively and efficiently inhibit the expression of an APOC3 gene.Further, there exists a need for compositions that include novelAPOC3-specific RNAi agents for the treatment of diseases associatedwith, among other things, elevated triglyceride (TG) levels.

In general, the present disclosure features APOC3 gene-specific RNAiagents, compositions that include APOC3 RNAi agents, and methods forinhibiting expression of an APOC3 gene in vitro and/or in vivo using theAPOC3 RNAi agents and compositions that include APOC3 RNAi agentsdescribed herein. The APOC3 RNAi agents disclosed herein can selectivelyand efficiently decrease or inhibit expression of an APOC3 gene, andthereby reduce TG levels and/or cholesterol levels in a subject, e.g., ahuman or animal subject.

The described APOC3 RNAi agents can be used in methods for therapeutictreatment (including the prophylactic and preventative treatment) ofsymptoms and diseases associated with elevated TG levels and/or elevatedcholesterol levels, including, but not limited to, obesity,hyperlipidemia, hypertriglyceridemia, abnormal lipid and/or cholesterolmetabolism, atherosclerosis, cardiovascular disease, coronary arterydisease, hypertriglyceridemia induced pancreatitis, metabolic syndrome,type II diabetes mellitus, familial chylomicronemia syndrome, familialpartial lipodystrophy, and other metabolic-related disorders anddiseases. The APOC3 RNAi agents disclosed herein can selectively reduceAPOC3 gene expression, which can lead to a reduction in, among otherthings, TG levels and/or cholesterol levels, in a subject. The methodsdisclosed herein include the administration of one or more APOC3 RNAiagents to a subject, e.g., a human or animal subject, using any suitablemethods known in the art, such as subcutaneous injection or intravenousadministration.

In one aspect, the disclosure features RNAi agents for inhibitingexpression of the human APOC3 gene, wherein the RNAi agent includes asense strand and an antisense strand. Also described herein arecompositions that include or consist of an RNAi agent capable ofinhibiting the expression of an APOC3 gene, wherein the APOC3 RNAi agentincludes or consists of a sense strand and an antisense strand, and thecomposition further includes at least one pharmaceutically acceptableexcipient. The compositions described herein that include one or more ofthe disclosed APOC3 RNAi agents are able to selectively and efficientlydecrease expression of an APOC3 gene. The compositions that include oneor more APOC3 RNAi agents can be administered to a subject, such as ahuman or animal subject, for the treatment (including prophylactictreatment or inhibition) of symptoms and diseases associated withelevated TG levels, elevated cholesterol, and/or enhanced APOC3expression.

An APOC3 RNAi agent described herein includes a sense strand (alsoreferred to as a passenger strand), and an antisense strand (alsoreferred to as a guide strand). The sense strand and the antisensestrand can be partially, substantially, or fully complementary to eachother. The length of the RNAi agent sense and antisense strandsdescribed herein each can be 16 to 30 nucleotides in length. In someembodiments, the sense and antisense strands are independently 17 to 26nucleotides in length. The sense and antisense strands can be either thesame length or different lengths. In some embodiments, the sense andantisense strands are independently 21 to 26 nucleotides in length. Insome embodiments, the sense and antisense strands are independently 21to 24 nucleotides in length. In some embodiments, both the sense strandand the antisense strand are 21 nucleotides in length. In someembodiments, the sense and/or antisense strands are independently 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides inlength. The RNAi agents described herein, upon delivery to a cellexpressing APOC3, inhibit the expression of one or more APOC3 genes invivo or in vitro.

A sense strand of the APOC3 RNAi agents described herein includes atleast 16 consecutive nucleotides that have at least 85% identity to acore stretch sequence (also referred to herein as a “core stretch” or“core sequence”) of the same number of nucleotides in an APOC3 mRNA. Insome embodiments, the sense strand core stretch having at least 85%identity to a sequence in an APOC3 mRNA is 16, 17, 18, 19, 20, 21, 22,or 23 nucleotides in length. In some embodiments, the sense strand corestretch having at least 85% identity to a sequence in an APOC3 mRNA is19 nucleotides in length. In some embodiments, this sense strand corestretch is 17 nucleotides in length.

An antisense strand of an APOC3 RNAi agent includes at least 16consecutive nucleotides that have at least 85% complementarity to a corestretch of the same number of nucleotides in an APOC3 mRNA and to a corestretch of the same number of nucleotides in the corresponding sensestrand. In some embodiments, the antisense strand core stretch having atleast 85% complementarity to a sequence in an APOC3 mRNA or thecorresponding sense strand is 16, 17, 18, 19, 20, 21, 22, or 23nucleotides in length. In some embodiments, this antisense strand corestretch is 19 nucleotides in length. In some embodiments, this antisensestrand core stretch is 17 nucleotides in length.

In some embodiments, the APOC3 RNAi agents disclosed herein target theportion of an APOC3 gene having the sequence of any of the sequencesdisclosed in Table 1.

Examples of APOC3 RNAi agent sense strands and antisense strands thatcan be included in the APOC3 RNAi agents disclosed herein are providedin Tables 3, 4, and 5. Examples of APOC3 RNAi agent duplexes areprovided in Tables 3 and 6. Examples of 19-nucleotide core stretchsequences that that consist of or are included in the sense strands andantisense strands of APOC3 RNAi agents disclosed herein, are provided inTable 2.

In another aspect, the disclosure features methods for delivering APOC3RNAi agents to liver cells in a subject, such as a mammal, in vivo. Alsodescribed herein are compositions for use in such methods. The one ormore APOC3 RNAi agents can be delivered to target cells or tissues usingany oligonucleotide delivery technology known in the art. Nucleic aciddelivery methods include, but are not limited to, by encapsulation inliposomes, by iontophoresis, or by incorporation into other vehicles,such as hydrogels, cyclodextrins, biodegradable nanocapsules, andbioadhesive microspheres, proteinaceous vectors, or DynamicPolyconjugates™ (DPCs) (see, for example WO 2000/053722, WO2008/0022309, WO 2011/104169, and WO 2012/083185, each of which isincorporated herein by reference).

In some embodiments, an APOC3 RNAi agent is delivered to target cells ortissues by covalently linking or conjugating the RNAi agent to atargeting group, such as an asialoglycoprotein receptor ligand. In someembodiments, an asialoglycoprotein receptor ligand includes, consistsof, or consists essentially of, a galactose or galactose derivativecluster. In some embodiments, an APOC3 RNAi agent is linked to atargeting ligand comprising the galactose derivativeN-acetyl-galactosamine. In some embodiments, a galactose derivativecluster includes an N-acetyl-galactosamine trimer or anN-acetyl-galactosamine tetramer. In some embodiments, a galactosederivative cluster is an N-acetyl-galactosamine trimer or anN-acetyl-galactosamine tetramer. In some embodiments, the APOC3 RNAiagents that are conjugated to targeting ligands that includeN-acetyl-galactosamine are selectively internalized by liver cells, andhepatocytes in particular, either through receptor-mediated endocytosisor by other means. Example targeting groups useful for delivering RNAiagents are disclosed, for example, in International Patent ApplicationPublication Nos. WO 2018/044350 and WO 2017/156012, which areincorporated herein by reference in their entirety.

A targeting group can be linked to the 3′ or 5′ end of a sense strand oran antisense strand of an APOC3 RNAi agent. In some embodiments, atargeting group is linked to the 3′ or 5′ end of the sense strand. Insome embodiments, a targeting group is linked to the 5′ end of the sensestrand. In some embodiments, a targeting group is linked internally to anucleotide on the sense strand and/or the antisense strand of the RNAiagent. In some embodiments, a targeting group is linked to the RNAiagent via a linker.

A targeting group, with or without a linker, can be linked to the 5′ or3′ end of any of the sense and/or antisense strands disclosed in Tables2, 3, 4 and 5. A linker, with or without a targeting group, can beattached to the 5′ or 3′ end of any of the sense and/or antisensestrands disclosed in Tables 2, 3, 4 and 5.

In some embodiments, described herein are compositions that include oneor more APOC3 RNAi agents having the duplex sequences disclosed in Table6.

In a further aspect, described herein are pharmaceutical compositionsthat include one or more described APOC3 RNAi agent(s), optionallycombined with one or more additional (i.e., second, third, etc.)therapeutics. In some embodiments, the pharmaceutical compositions thatinclude one or more described APOC3 RNAi agent(s), optionally combinedwith one or more additional (i.e., second, third, etc.) therapeutics,can be formulated in a pharmaceutically acceptable carrier or diluent.In some embodiments, these compositions can be administered to asubject, such as a mammal. In some embodiments, the mammal is a human.

In some embodiments, the compositions described herein include acombination or cocktail of at least two APOC3 RNAi agents havingdifferent nucleotide sequences. In some embodiments, the two or moredifferent APOC3 RNAi agents are each separately and independently linkedto targeting groups. In some embodiments, the two or more differentAPOC3 RNAi agents are each linked to targeting groups that include orconsist of targeting ligands that include one or more moieties thattarget the asialoglycoprotein receptor. In some embodiments, the two ormore different APOC3 RNAi agents are each linked to targeting groupsthat include or consist of targeting ligands that include one or moregalactose derivatives. In some embodiments, the two or more differentAPOC3 RNAi agents are each linked to targeting groups that include orconsist of targeting ligands that include one or moreN-acetyl-galactosamines.

In another aspect, the disclosure features methods for inhibiting APOC3gene expression in a subject, wherein the methods include administeringto a subject or to a cell of a subject an amount of an APOC3 RNAi agentcapable of inhibiting the expression of an APOC3 gene, wherein the APOC3RNAi agent comprises a sense strand and an antisense strand, and whereinthe antisense strand includes the sequence of any one of the antisensestrand nucleotide sequences in Table 2, Table 3, or Table 4. In someembodiments, compositions for delivering an APOC3 RNAi agent to a livercell, particularly hepatocytes, in vivo are described, the compositionscomprising: an APOC3 RNAi agent conjugated to a targeting group. In someembodiments, the targeting group is an asialoglycoprotein receptorligand.

In some embodiments, disclosed herein are methods of inhibitingexpression of an APOC3 gene, wherein the methods include administeringto a subject or to a cell of a subject an amount of an APOC3 RNAi agentcapable of inhibiting the expression of an APOC3 gene, wherein the APOC3RNAi agent comprises a sense strand and an antisense strand, and whereinthe sense strand includes the sequence of any one of the sense strandnucleotide sequences in Table 2, Table 3, or Table 5. Also describedherein are compositions for use in such methods.

In a further aspect, the disclosure features methods of treatment(including preventative or prophylactic treatment) of diseases orsymptoms caused by elevated TG levels and/or elevated cholesterollevels, wherein the methods include administering to a subject in needthereof an APOC3 RNAi agent having an antisense strand that includes thesequence of any of the sequences in Tables 2, 3, or 4. In someembodiments, described herein are methods of treatment (includingpreventative or prophylactic treatment) of diseases or symptoms causedby elevated TG levels and/or elevated cholesterol levels, wherein themethods include administering to a subject in need thereof an APOC3 RNAiagent having a sense strand comprising the sequence of any of thesequences in Tables 2, 3, or 5. Also described herein are compositionsfor use in such methods.

Also described are methods of treating a human subject having apathological state (such as a condition or disease), or being at risk ofdeveloping a pathological state, that is mediated at least in part byAPOC3 gene expression, the methods comprising the step of administeringto the subject a therapeutically effective amount of an APOC3 RNAi agentand/or APOC3 RNAi agent-containing composition. The method of treating asubject with an APOC3 RNAi agent and/or APOC3 RNAi agent-containingcomposition can optionally be combined with one or more steps ofadministering one or more additional (i.e., second, third, etc.)therapeutics or treatments. The APOC3 RNAi agent and additionaltherapeutics can be administered in a single composition or they can beadministered separately. An additional therapeutic can be another APOC3RNAi agent (e.g., an APOC3 RNAi agent that targets a different sequencewithin the APOC3 gene). An additional therapeutic can also be a smallmolecule drug, antibody, antibody fragment, and/or aptamer. In someembodiments, the one or more additional therapeutics is a statin, suchas atorvastatin, fluvastatin, pravastatin, pitavastatin, rosuvastatin,or simvastatin.

In some embodiments, the described APOC3 RNAi agent(s) are optionallycombined with one or more additional therapeutics, wherein the one ormore additional therapeutics is administered separately in separatedosage forms from the RNAi agent (e.g., the APOC3 RNAi agent isadministered by subcutaneous injection, while the additional therapeuticinvolved in the method of treatment dosing regimen is administeredorally). In some embodiments, the described APOC3 RNAi agent(s) areadministered to a subject in need thereof via subcutaneous injection,and the one or more optional additional therapeutics are administeredorally, which together provide for a treatment regimen for diseases andconditions associated with elevated TG and/or cholesterol levels. Insome embodiments, the described APOC3 RNAi agent(s) are administered toa subject in need thereof via subcutaneous injection, and the one ormore optional additional therapeutics are administered via a separatesubcutaneous injection. In some embodiments, the APOC3 RNAi agent andone or more additional therapeutics are combined into a single dosageform (e.g., a “cocktail” formulated into a single composition forsubcutaneous injection). The APOC3 RNAi agents, with or without the oneor more additional therapeutics, can be combined with one or moreexcipients to form pharmaceutical compositions.

In some embodiments, disclosed herein are methods for inhibitingexpression of an APOC3 gene, the methods include administering to thecell or subject an APOC3 RNAi agent that includes a sense strandcomprising, consisting of, or consisting essentially of the sequence ofany of the sequences in Tables 2, 3, or 5. In some embodiments,disclosed herein are methods of inhibiting expression of an APOC3 gene,wherein the methods include administering an APOC3 RNAi agent thatincludes a sense strand comprising the sequence of any of the sequencesin Table 5, and the antisense strand comprising, consisting of, orconsisting essentially of the sequence of any of the sequences in Table4.

In some embodiments, disclosed herein are methods of inhibitingexpression of an APOC3 gene in a cell or a subject, wherein the methodsinclude administering to the cell or subject an APOC3 RNAi agent thatincludes a sense strand that includes the nucleobase sequence of any ofthe sequences in Table 5, and an antisense strand that includes thenucleobase sequence of any of the sequences in Table 4. In otherembodiments, disclosed herein are methods of inhibiting expression of anAPOC3 gene, wherein the methods include administering to a subject anAPOC3 RNAi agent that includes a sense strand consisting of the modifiedsequence of any of the modified sequences in Table 5, and the antisensestrand consisting of the modified sequence of any of the modifiedsequences in Table 4.

In some embodiments, compositions for delivering an APOC3 RNAi agent toa liver cell, particularly hepatocytes, in vivo, are described, thecompositions comprising: an APOC3 RNAi agent conjugated to a targetinggroup. In some embodiments, the targeting group is an asialoglycoproteinreceptor ligand (i.e., a ligand that includes a compound having affinityfor the asialoglycoprotein receptor). In some embodiments, the targetinggroup comprises N-acetyl-galactosamine.

In some embodiments, disclosed herein are methods for inhibitingexpression of an APOC3 gene in a cell, the methods include administeringone or more APOC3 RNAi agents having the duplex structure of a duplexset forth in Table 6.

In some embodiments, disclosed herein are methods of treatment(including prophylactic or preventative treatment) of diseases,disorders, or symptoms caused by elevated TG levels and/or elevatedcholesterol levels, wherein the methods include administering to asubject in need thereof a therapeutically effective amount of an APOC3RNAi agent that includes an antisense strand that is at least partiallycomplementary to the portion of the APOC3 mRNA having the sequence inTable 1. In some embodiments, disclosed herein are methods of treatment(including prophylactic or preventative treatment) of diseases orsymptoms caused by elevated TG levels and/or elevated cholesterollevels, wherein the methods include administering to a subject in needthereof a therapeutically effective amount of an APOC3 RNAi agent thatincludes an antisense strand comprising the sequence of any of thesequences in Tables 2, 3, or 4, and a sense strand that comprises any ofthe sequences in Tables 2, 3, or 5 that is at least partiallycomplementary to the antisense strand. In some embodiments, disclosedherein are methods of treatment (including prophylactic or preventativetreatment) of diseases or symptoms caused by elevated TG levels and/orelevated cholesterol levels, wherein the methods include administeringto a subject in need thereof a therapeutically effective amount of anAPOC3 RNAi agent that includes a sense strand that comprises any of thesequences in Tables 2, 3, or 5, and an antisense strand comprising thesequence of any of the sequences in Tables 2, 3, or 4 that is at leastpartially complementary to the sense strand.

In some embodiments, disclosed herein are methods for inhibitingexpression of an APOC3 gene in a cell, wherein the methods includeadministering to the cell an APOC3 RNAi agent that includes an antisensestrand that is at least partially complementary to the portion of theAPOC3 mRNA having the sequence in Table 1. In some embodiments,disclosed herein are methods of inhibiting expression of an APOC3 genein a cell, wherein the methods include administering to a cell an APOC3RNAi agent that includes an antisense strand comprising the sequence ofany of the sequences in Tables 2, 3, or 4, and a sense strand thatcomprises any of the sequences in Tables 2, 3, or 5 that is at leastpartially complementary to the antisense strand. In some embodiments,disclosed herein are methods of inhibiting expression of an APOC3 genein a cell, wherein the methods include administering an APOC3 RNAi agentthat includes a sense strand that comprises any of the sequences inTables 2, 3, or 5, and an antisense strand that includes the sequence ofany of the sequences in Tables 2, 3, or 4 that is at least partiallycomplementary to the sense strand.

In some embodiments, disclosed herein are compositions for inhibitingexpression of an APOC3 gene in a cell, wherein the methods includeadministering a composition that comprises an APOC3 RNAi agent havingthe duplex structure of a duplex set forth in Table 6.

In some embodiments, disclosed herein are compositions for delivering anAPOC3 RNAi agent to a liver cell in vivo, the composition including anAPOC3 RNAi agent conjugated or linked to a targeting group. In someembodiments, the targeting group is an asialoglycoprotein receptorligand. In some embodiments, compositions for delivering an APOC3 RNAiagent to a liver cell in vivo are described, the composition includingan APOC3 RNAi agent linked to an N-acetyl-galactosamine targetingligand.

The APOC3 RNAi agents disclosed herein are designed to target specificpositions on an APOC3 gene (SEQ ID NO: 1). As defined herein, anantisense strand sequence is designed to target an APOC3 gene at a givenposition on the gene when the 5′ terminal nucleobase of the antisensestrand would be aligned with the position that is 19 nucleotidesdownstream (towards the 3′ end) from the position on the gene when basepairing to the gene. For example, as illustrated in Tables 1 and 2herein, an antisense strand sequence designed to target an APOC3 gene atposition 438 requires that when base pairing to the gene, the 5′terminal nucleobase of the antisense strand is aligned with position 456of the APOC3 gene.

As provided herein, an APOC3 RNAi agent does not require that thenucleobase at position 1 (5′→3′) of the antisense strand becomplementary to the gene, provided that there is at least 85%complementarity (e.g., at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, or 100% complementarity) of the antisense strand andthe gene across a core stretch sequence of at least 16 consecutivenucleotides. For example, for an APOC3 RNAi agent disclosed herein thatis designed to target position 438 of an APOC3 gene, the 5′ terminalnucleobase of the antisense strand of the of the APOC3 RNAi agent mustbe aligned with position 456 of the gene; however, the 5′ terminalnucleobase of the antisense strand may be, but is not required to be,complementary to position 456 of an APOC3 gene, provided that there isat least 85% complementarity (e.g., at least 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, or 100% complementarity) of theantisense strand and the gene across a core stretch sequence of at least16 consecutive nucleotides. As shown by, among other things, the variousexamples disclosed herein, the specific site of binding of the gene bythe antisense strand of the APOC3 RNAi agent (e.g., whether the APOC3RNAi agent is designed to target an APOC3 gene at position 438, atposition 506, at position 432, or at some other position) is importantto the level of inhibition achieved by the APOC3 RNAi agent.

The use of APOC3 RNAi agents provides methods for therapeutic (includingprophylactic) treatment of diseases/disorders associated with elevatedTG and/or cholesterol levels and/or enhanced or elevated APOC3expression. The described APOC3 RNAi agents mediate RNA interference toinhibit the expression of one or more genes necessary for production ofAPOC3. APOC3 RNAi agents can also be used to treat or prevent variousdiseases or disorders, including obesity, hyperlipidemia,hypertriglyceridemia, abnormal lipid and/or cholesterol metabolism,atherosclerosis, cardiovascular disease, coronary artery disease,hypertriglyceridemia mediated pancreatitis, metabolic syndrome, type 11diabetes mellitus, familial chylomicronemia syndrome, familial partiallipodystrophy, and other metabolic-related disorders and diseases.Furthermore, compositions for delivery of APOC3 RNAi agents to livercells in vivo are described.

The pharmaceutical compositions including one or more APOC3 RNAi agentscan be administered in a number of ways depending upon whether local orsystemic treatment is desired. Administration can be, but is not limitedto, intravenous, intraarterial, subcutaneous, intraperitoneal, subdermal(e.g., via an implanted device), and intraparenchymal administration. Insome embodiments, the pharmaceutical compositions described herein areadministered by subcutaneous injection.

In some embodiments, disclosed herein are compositions for delivering anAPOC3 RNAi agent to a liver cell in vivo, wherein the compositionincludes an APOC3 RNAi agent conjugated or linked to a targeting group.In some embodiments, the targeting group is an asialoglycoproteinreceptor ligand. In some embodiments, compositions for delivering anAPOC3 RNAi agent to a liver cell in vivo are described, wherein thecomposition includes an APOC3 RNAi agent linked to a targeting ligandthat includes N-acetyl-galactosamine.

In some embodiments, the APOC3 RNAi agents described herein can includeone or more targeting groups having the structure of (NAG25), (NAG25)s,(NAG26), (NAG26)s, (NAG27), (NAG27)s, (NAG28), (NAG28)s, (NAG29),(NAG29)s, (NAG30), (NAG30)s, (NAG31), (NAG31)s, (NAG32), (NAG32)s,(NAG33), (NAG33)s, (NAG34), (NAG34)s, (NAG35), (NAG35)s, (NAG36),(NAG36)s, (NAG37), (NAG37)s, (NAG38), (NAG38)s, (NAG39), (NAG39)s, eachas defined herein in Table 7.

In some embodiments, the APOC3 RNAi agents described herein include onetargeting group at the 5′ end of the sense strand having the structureof (NAG25), (NAG25)s, (NAG26), (NAG26)s, (NAG27), (NAG27)s, (NAG28),(NAG28)s, (NAG29), (NAG29)s, (NAG30), (NAG30)s, (NAG31), (NAG31)s,(NAG32), (NAG32)s, (NAG33), (NAG33)s, (NAG34), (NAG34)s, (NAG35),(NAG35)s, (NAG36), (NAG36)s, (NAG37), (NAG37)s, (NAG38), (NAG38)s,(NAG39), (NAG39)s, each as defined herein in Table 7.

The described APOC3 RNAi agents and/or compositions that include APOC3RNAi agents can be used in methods for therapeutic treatment of diseasesor conditions caused by elevated TG levels. Such methods includeadministration of an APOC3 RNAi agent as described herein to a subject,e.g., a human or animal subject. In some embodiments, one or more of thedescribed APOC3 RNAi agents are administered to a subject, such as amammal, in a pharmaceutically acceptable carrier or diluent. In someembodiments, the mammal is a human.

The APOC3 RNAi agents disclosed herein can be incorporated into acomposition comprising one or more disclosed APOC3 RNAi agent and atleast one pharmaceutically acceptable excipient. In some embodiments,the compositions disclosed herein comprising one or more of thedisclosed APOC3 RNAi agents and at least one pharmaceutically acceptableexcipient is a pharmaceutical composition.

In some embodiments, the compositions comprising one or more disclosedAPOC3 RNAi agents and at least one pharmaceutically acceptable excipientcan further comprise one or more additional therapeutics or treatments.

In some embodiments, the compositions described herein comprising one ormore APOC3 RNAi agents are packaged in a kit, container, pack,dispenser, pre-filled syringes, or vials. In some embodiments, thecompositions described herein are administered parenterally.

In some embodiments, an APOC3 RNAi agent disclosed herein includes anantisense strand that consists of, consists essentially of, or comprisesa nucleobase sequence differing by 0 or 1 nucleobases from thenucleotide sequence (5′→3′) UCACUGAGAAUACUGUCCCUC (SEQ ID NO:3). In someembodiments, an APOC3 RNAi agent disclosed herein includes an antisensestrand that consists of, consists essentially of, or comprises anucleotide sequence differing by no more than 1 nucleotide from thenucleotide sequence (5′→3′) UCACUGAGAAUACUGUCCCUC (SEQ ID NO:3), whereinall or substantially all of the nucleotides are modified nucleotides. Insome embodiments, an APOC3 RNAi agent disclosed herein includes anantisense strand that consists of, consists essentially of, or comprisesa nucleobase sequence differing by 0 or 1 nucleobases from thenucleotide sequence (5′→3′) UCACUGAGAAUACUGUCCCUC (SEQ ID NO:3), whereinSEQ ID NO:3 is located at positions 1-21 (5′→3′) of the antisensestrand.

In some embodiments, an APOC3 RNAi agent disclosed herein includes anantisense strand that consists of, consists essentially of, or comprisesa modified nucleotide sequence differing by no more than 1 nucleotidefrom the nucleotide sequence (5′→3′) usCfsasCfuGfagaauAfcUfgUfcCfcUfsc(SEQ ID NO:2), wherein a, c, g, and u represent 2′-O-methyl adenosine,cytidine, guanosine, or uridine, respectively; Af, Cf, Gf, and Ufrepresent 2′-fluoro adenosine, cytidine, guanosine, or uridine,respectively; and s represents a phosphorothioate linkage, and whereinthe sense strand is at least substantially complementary to theantisense strand. As the person of ordinary skill in the art wouldclearly understand, the inclusion of a phosphorothioate linkage as shownin the modified nucleotide sequences disclosed herein replaces thephosphodiester linkage typically present in oligonucleotides (see, e.g.,FIGS. 1A through 1I showing all internucleoside linkages). In someembodiments, an APOC3 RNAi agent disclosed herein includes an antisensestrand that consists of, consists essentially of, or comprises thenucleotide sequence (5′→3′) usCfsasCfuGfagaauAfcUfgUfcCfcUfsc (SEQ IDNO:2), wherein a, c, g, and u represent 2′-O-methyl adenosine, cytidine,guanosine, or uridine, respectively; Af, Cf, Gf, and Uf represent2′-fluoro adenosine, cytidine, guanosine, or uridine, respectively; ands represents a phosphorothioate linkage, and wherein the sense strand isat least substantially complementary to the antisense strand.

In some embodiments, an APOC3 RNAi agent disclosed herein includes anantisense strand that consists of, consists essentially of, or comprisesa nucleobase sequence differing by 0 or 1 nucleobases from thenucleotide sequence (5′→3′) UCACUGAGAAUACUGUCCCGU (SEQ ID NO:5). In someembodiments, an APOC3 RNAi agent disclosed herein includes an antisensestrand that consists of, consists essentially of, or comprises anucleotide sequence differing by no more than 1 nucleotide from thenucleotide sequence (5′→3′) UCACUGAGAAUACUGUCCCGU (SEQ ID NO:5), whereinall or substantially all of the nucleotides are modified nucleotides. Insome embodiments, an APOC3 RNAi agent disclosed herein includes anantisense strand that consists of, consists essentially of, or comprisesa nucleobase sequence differing by 0 or 1 nucleobases from thenucleotide sequence (5′→3′) UCACUGAGAAUACUGUCCCGU (SEQ ID NO:5), whereinSEQ ID NO:5 is located at positions 1-21 (5′→3′) of the antisensestrand.

In some embodiments, an APOC3 RNAi agent disclosed herein includes anantisense strand that consists of, consists essentially of, or comprisesa modified nucleotide sequence differing by no more than 1 nucleotidefrom the nucleotide sequence (5′→3′) usCfsasCfuGfagaauAfcUfgUfcCfcGfsu(SEQ ID NO:4), wherein a, c, g, and u represent 2′-O-methyl adenosine,cytidine, guanosine, or uridine, respectively; Af, Cf, Gf, and Ufrepresent 2′-fluoro adenosine, cytidine, guanosine, or uridine,respectively; and s represents a phosphorothioate linkage, and whereinthe sense strand is at least substantially complementary to theantisense strand. As the person of ordinary skill in the art wouldclearly understand, the inclusion of a phosphorothioate linkage as shownin the modified nucleotide sequences disclosed herein replaces thephosphodiester linkage typically present in oligonucleotides (see, e.g.,FIGS. 1A through 1I showing all internucleoside linkages). In someembodiments, an APOC3 RNAi agent disclosed herein includes an antisensestrand that consists of, consists essentially of, or comprises thenucleotide sequence (5′→3′) usCfsasCfuGfagaauAfcUfgUfcCfcGfsu (SEQ IDNO:4), wherein a, c, g, and u represent 2′-O-methyl adenosine, cytidine,guanosine, or uridine, respectively; Af, Cf, Gf, and Uf represent2′-fluoro adenosine, cytidine, guanosine, or uridine, respectively; ands represents a phosphorothioate linkage, and wherein the sense strand isat least substantially complementary to the antisense strand.

In some embodiments, an APOC3 RNAi agent disclosed herein includes anantisense strand that consists of, consists essentially of, or comprisesa modified nucleotide sequence differing by no more than 1 nucleotidefrom the nucleotide sequence (5′→3′) usCfsascugagaauAfcUfgUfcCfcUfsc(SEQ ID NO:6), wherein a, c, g, and u represent 2′-O-methyl adenosine,cytidine, guanosine, or uridine, respectively; Af, Cf, Gf, and Ufrepresent 2′-fluoro adenosine, cytidine, guanosine, or uridine,respectively; and s represents a phosphorothioate linkage, and whereinthe sense strand is at least substantially complementary to theantisense strand. As the person of ordinary skill in the art wouldclearly understand, the inclusion of a phosphorothioate linkage as shownin the modified nucleotide sequences disclosed herein replaces thephosphodiester linkage typically present in oligonucleotides (see, e.g.,FIGS. 1A through 11I showing all internucleoside linkages). In someembodiments, an APOC3 RNAi agent disclosed herein includes an antisensestrand that consists of, consists essentially of, or comprises thenucleotide sequence (5′→3′) usCfsascugagaauAfcUfgUfcCfcUfsc (SEQ IDNO:6), wherein a, c, g, and u represent 2′-O-methyl adenosine, cytidine,guanosine, or uridine, respectively; Af, Cf, Gf, and Uf represent2′-fluoro adenosine, cytidine, guanosine, or uridine, respectively; ands represents a phosphorothioate linkage, and wherein the sense strand isat least substantially complementary to the antisense strand.

In some embodiments, an APOC3 RNAi agent disclosed herein includes anantisense strand that consists of, consists essentially of, or comprisesa nucleobase sequence differing by 0 or 1 nucleobases from thenucleotide sequence (5′→3′) UUCUUGUCCAGCUUUAUUGGC (SEQ ID NO:8). In someembodiments, an APOC3 RNAi agent disclosed herein includes an antisensestrand that consists of, consists essentially of, or comprises anucleotide sequence differing by no more than 1 nucleotide from thenucleotide sequence (5′→3′) UUCUUGUCCAGCUUUAUUGGC (SEQ ID NO:8), whereinall or substantially all of the nucleotides are modified nucleotides. Insome embodiments, an APOC3 RNAi agent disclosed herein includes anantisense strand that consists of, consists essentially of, or comprisesa nucleobase sequence differing by 0 or 1 nucleobases from thenucleotide sequence (5′→3′) UUCUUGUCCAGCUUUAUUGGC (SEQ ID NO:8), whereinSEQ ID NO:8 is located at positions 1-21 (5′→3′) of the antisensestrand.

In some embodiments, an APOC3 RNAi agent disclosed herein includes anantisense strand that consists of, consists essentially of, or comprisesa modified nucleotide sequence differing by no more than 1 nucleotidefrom the nucleotide sequence (5′→3′) usUfscsUfuGfuCfcAfgCfuUfuAfuUfgGfsc(SEQ ID NO:7), wherein a, c, g, and u represent 2′-O-methyl adenosine,cytidine, guanosine, or uridine, respectively; Af, Cf, Gf, and Ufrepresent 2′-fluoro adenosine, cytidine, guanosine, or uridine,respectively; and s represents a phosphorothioate linkage, and whereinthe sense strand is at least substantially complementary to theantisense strand. In some embodiments, an APOC3 RNAi agent disclosedherein includes an antisense strand that consists of, consistsessentially of, or comprises the nucleotide sequence (5′→3′)usUfscsUfuGfuCfcAfgCfuUfuAfuUfgGfsc (SEQ ID NO:7), wherein a, c, g, andu represent 2′-O-methyl adenosine, cytidine, guanosine, or uridine,respectively; Af, Cf, Gf, and Uf represent 2′-fluoro adenosine,cytidine, guanosine, or uridine, respectively; and s represents aphosphorothioate linkage, and wherein the sense strand is at leastsubstantially complementary to the antisense strand.

In some embodiments, an APOC3 RNAi agent disclosed herein includes anantisense strand that consists of, consists essentially of, or comprisesa nucleobase sequence differing by 0 or 1 nucleobases from thenucleotide sequence (5′→3′) AGAAUACUGUCCCUUUUAGGG (SEQ ID NO: 10). Insome embodiments, an APOC3 RNAi agent disclosed herein includes anantisense strand that consists of, consists essentially of, or comprisesa nucleotide sequence differing by no more than 1 nucleotide from thenucleotide sequence (5′→3′) AGAAUACUGUCCCUUUUAGGG (SEQ ID NO:10),wherein all or substantially all of the nucleotides are modifiednucleotides. In some embodiments, an APOC3 RNAi agent disclosed hereinincludes an antisense strand that consists of, consists essentially of,or comprises a nucleobase sequence differing by 0 or 1 nucleobases fromthe nucleotide sequence (5′→3′) AGAAUACUGUCCCUUUUAGGG (SEQ ID NO:10),wherein SEQ ID NO:10 is located at positions 1-21 (5′→3′) of theantisense strand.

In some embodiments, an APOC3 RNAi agent disclosed herein includes anantisense strand that consists of, consists essentially of, or comprisesa modified nucleotide sequence differing by no more than 1 nucleotidefrom the nucleotide sequence (5′→3′) asGfsasAfuAfcUfgUfcCfcUfuUfuAfgGfsg(SEQ ID NO:9), wherein a, c, g, and u represent 2′-O-methyl adenosine,cytidine, guanosine, or uridine, respectively; Af, Cf, Gf, and Ufrepresent 2′-fluoro adenosine, cytidine, guanosine, or uridine,respectively; and s represents a phosphorothioate linkage, and whereinthe sense strand is at least substantially complementary to theantisense strand. In some embodiments, an APOC3 RNAi agent disclosedherein includes an antisense strand that consists of, consistsessentially of, or comprises the nucleotide sequence (5′→3′)asGfsasAfuAfcUfgUfcCfcUfuUfuAfgGfsg (SEQ ID NO:9), wherein a, c, g, andu represent 2′-O-methyl adenosine, cytidine, guanosine, or uridine,respectively; Af, Cf, Gf, and Uf represent 2′-fluoro adenosine,cytidine, guanosine, or uridine, respectively; and s represents aphosphorothioate linkage, and wherein the sense strand is at leastsubstantially complementary to the antisense strand.

In some embodiments, an APOC3 RNAi agent disclosed herein includes anantisense strand that consists of, consists essentially of, or comprisesa nucleobase sequence differing by 0 or 1 nucleobases from thenucleotide sequence (5′→3′) AGAAUACUGUCCCUUUUAAGC (SEQ ID NO: 12). Insome embodiments, an APOC3 RNAi agent disclosed herein includes anantisense strand that consists of, consists essentially of, or comprisesa nucleotide sequence differing by no more than 1 nucleotide from thenucleotide sequence (5′→3′) AGAAUACUGUCCCUUUUAAGC (SEQ ID NO: 12),wherein all or substantially all of the nucleotides are modifiednucleotides. In some embodiments, an APOC3 RNAi agent disclosed hereinincludes an antisense strand that consists of, consists essentially of,or comprises a nucleobase sequence differing by 0 or 1 nucleobases fromthe nucleotide sequence (5′→3′) AGAAUACUGUCCCUUUUAAGC (SEQ ID NO: 12),wherein SEQ ID NO: 12 is located at positions 1-21 (5′→3′) of theantisense strand.

In some embodiments, an APOC3 RNAi agent disclosed herein includes anantisense strand that consists of, consists essentially of, or comprisesa modified nucleotide sequence differing by no more than 1 nucleotidefrom the nucleotide sequence (5′→3′) asGfsasAfuAfcUfgUfcCfcUfuUfuAfaGfsc(SEQ ID NO: 11), wherein a, c, g, and u represent 2′-O-methyl adenosine,cytidine, guanosine, or uridine, respectively; Af, Cf, Gf, and Ufrepresent 2′-fluoro adenosine, cytidine, guanosine, or uridine,respectively; and s represents a phosphorothioate linkage, and whereinthe sense strand is at least substantially complementary to theantisense strand. In some embodiments, an APOC3 RNAi agent disclosedherein includes an antisense strand that consists of, consistsessentially of, or comprises the nucleotide sequence (5′→3′)asGfsasAfuAfcUfgUfcCfcUfuUfuAfaGfsc (SEQ ID NO: 11), wherein a, c, g,and u represent 2′-O-methyl adenosine, cytidine, guanosine, or uridine,respectively; Af, Cf, Gf, and Uf represent 2′-fluoro adenosine,cytidine, guanosine, or uridine, respectively; and s represents aphosphorothioate linkage, and wherein the sense strand is at leastsubstantially complementary to the antisense strand.

In some embodiments, an APOC3 RNAi agent disclosed herein includes anantisense strand that consists of, consists essentially of, or comprisesa nucleobase sequence differing by 0 or 1 nucleobases from thenucleotide sequence (5′→3′) UGAGAAUACUGUCCCUUUGCC (SEQ ID NO: 14). Insome embodiments, an APOC3 RNAi agent disclosed herein includes anantisense strand that consists of, consists essentially of, or comprisesa nucleotide sequence differing by no more than 1 nucleotide from thenucleotide sequence (5′→3′) UGAGAAUACUGUCCCUUUGCC (SEQ ID NO: 14),wherein all or substantially all of the nucleotides are modifiednucleotides. In some embodiments, an APOC3 RNAi agent disclosed hereinincludes an antisense strand that consists of, consists essentially of,or comprises a nucleobase sequence differing by 0 or 1 nucleobases fromthe nucleotide sequence (5′→3′) UGAGAAUACUGUCCCUUUGCC (SEQ ID NO:14),wherein SEQ ID NO:14 is located at positions 1-21 (5′→3′) of theantisense strand.

In some embodiments, an APOC3 RNAi agent disclosed herein includes anantisense strand that consists of, consists essentially of, or comprisesa modified nucleotide sequence differing by no more than 1 nucleotidefrom the nucleotide sequence (5′→3′) usGfsasGfaAfuAfcUfgUfcCfcUfuUfgcsc(SEQ ID NO: 13), wherein a, c, g, and u represent 2′-O-methyl adenosine,cytidine, guanosine, or uridine, respectively; Af, Cf, Gf, and Ufrepresent 2′-fluoro adenosine, cytidine, guanosine, or uridine,respectively; and s represents a phosphorothioate linkage, and whereinthe sense strand is at least substantially complementary to theantisense strand. In some embodiments, an APOC3 RNAi agent disclosedherein includes an antisense strand that consists of, consistsessentially of, or comprises the nucleotide sequence (5′→3′)usGfsasGfaAfuAfcUfgUfcCfcUfuUfgcsc (SEQ ID NO: 13), wherein a, c, g, andu represent 2′-O-methyl adenosine, cytidine, guanosine, or uridine,respectively; Af, Cf, Gf, and Uf represent 2′-fluoro adenosine,cytidine, guanosine, or uridine, respectively; and s represents aphosphorothioate linkage, and wherein the sense strand is at leastsubstantially complementary to the antisense strand.

In some embodiments, an APOC3 RNAi agent disclosed herein includes anantisense strand that consists of, consists essentially of, or comprisesa nucleobase sequence differing by 0 or 1 nucleobases from thenucleotide sequence (5′→3′) UCACUGAGAAUACUGUCCCUC (SEQ ID NO:3) and asense strand that consists of, consists essentially of, or comprises anucleobase sequence differing by 0 or 1 nucleobases from the nucleotidesequence (5′→3′) GAGGGACAGUAUUCUCAGUIA (SEQ ID NO: 16). (I represents aninosine nucleotide.) In some embodiments, an APOC3 RNAi agent disclosedherein includes an antisense strand that consists of, consistsessentially of, or comprises a nucleotide sequence differing by no morethan 1 nucleotide from the nucleotide sequence (5′→3′)UCACUGAGAAUACUGUCCCUC (SEQ ID NO:3), wherein all or substantially all ofthe nucleotides are modified nucleotides, and a sense strand thatconsists of, consists essentially of, or comprises a nucleotide sequencediffering by no more than 1 nucleotide from the nucleotide sequence(5′→3′) GAGGGACAGUAUUCUCAGUIA (SEQ ID NO:16), wherein all orsubstantially all of the nucleotides are modified nucleotides.

In some embodiments, an APOC3 RNAi agent disclosed herein includes anantisense strand that consists of, consists essentially of, or comprisesa nucleobase sequence differing by 0 or 1 nucleobases from thenucleotide sequence (5′→3′) UCACUGAGAAUACUGUCCCGU (SEQ ID NO:5) and asense strand that consists of, consists essentially of, or comprises anucleobase sequence differing by 0 or 1 nucleobases from the nucleotidesequence (5′→3′) ACGGGACAGUAUUCUCAGUIA (SEQ ID NO: 18). (I represents aninosine nucleotide.) In some embodiments, an APOC3 RNAi agent disclosedherein includes an antisense strand that consists of, consistsessentially of, or comprises a nucleotide sequence differing by no morethan 1 nucleotide from the nucleotide sequence (5′→3′)UCACUGAGAAUACUGUCCCGU (SEQ ID NO:5), wherein all or substantially all ofthe nucleotides are modified nucleotides, and a sense strand thatconsists of, consists essentially of, or comprises a nucleotide sequencediffering by no more than 1 nucleotide from the nucleotide sequence(5′→3′) ACGGGACAGUAUUCUCAGUIA (SEQ ID NO: 18), wherein all orsubstantially all of the nucleotides are modified nucleotides.

In some embodiments, an APOC3 RNAi agent disclosed herein includes anantisense strand that consists of, consists essentially of, or comprisesa nucleobase sequence differing by 0 or 1 nucleobases from thenucleotide sequence (5′→3′) UCACUGAGAAUACUGUCCCUC (SEQ ID NO:3) and asense strand that consists of, consists essentially of, or comprises anucleobase sequence differing by 0 or 1 nucleobases from the nucleotidesequence (5′→3′) GAGGGACAGUAUUCUCAGUGA (SEQ ID NO:21). In someembodiments, an APOC3 RNAi agent disclosed herein includes an antisensestrand that consists of, consists essentially of, or comprises anucleotide sequence differing by no more than 1 nucleotide from thenucleotide sequence (5′→3′) UCACUGAGAAUACUGUCCCUC (SEQ ID NO:3), whereinall or substantially all of the nucleotides are modified nucleotides,and a sense strand that consists of, consists essentially of, orcomprises a nucleotide sequence differing by no more than 1 nucleotidefrom the nucleotide sequence (5′→3′) GAGGGACAGUAUUCUCAGUGA (SEQ IDNO:21), wherein all or substantially all of the nucleotides are modifiednucleotides.

In some embodiments, an APOC3 RNAi agent disclosed herein includes anantisense strand that consists of, consists essentially of, or comprisesa nucleobase sequence differing by 0 or 1 nucleobases from thenucleotide sequence (5′→3′) UUCUUGUCCAGCUUUAUUGGC (SEQ ID NO:8) and asense strand that consists of, consists essentially of, or comprises anucleobase sequence differing by 0 or 1 nucleobases from the nucleotidesequence (5′→3′) GCCAAUAAAGCUGGACAAGAA (SEQ ID NO:23). In someembodiments, an APOC3 RNAi agent disclosed herein includes an antisensestrand that consists of, consists essentially of, or comprises anucleotide sequence differing by no more than 1 nucleotide from thenucleotide sequence (5′→3′) UUCUUGUCCAGCUUUAUUGGC (SEQ ID NO:8), whereinall or substantially all of the nucleotides are modified nucleotides,and a sense strand that consists of, consists essentially of, orcomprises a nucleotide sequence differing by no more than 1 nucleotidefrom the nucleotide sequence (5′→3′) GCCAAUAAAGCUGGACAAGAA (SEQ IDNO:23), wherein all or substantially all of the nucleotides are modifiednucleotides.

In some embodiments, an APOC3 RNAi agent disclosed herein includes anantisense strand that consists of, consists essentially of, or comprisesa nucleobase sequence differing by 0 or 1 nucleobases from thenucleotide sequence (5′→3′) UUCUUGUCCAGCUUUAUUGGC (SEQ ID NO:8) and asense strand that consists of, consists essentially of, or comprises anucleobase sequence differing by 0 or 1 nucleobases from the nucleotidesequence (5′→3′) GCCAAUAAAICUGGACAAGAA (SEQ ID NO:25). In someembodiments, an APOC3 RNAi agent disclosed herein includes an antisensestrand that consists of, consists essentially of, or comprises anucleotide sequence differing by no more than 1 nucleotide from thenucleotide sequence (5′→3′) UUCUUGUCCAGCUUUAUUGGC (SEQ ID NO:8), whereinall or substantially all of the nucleotides are modified nucleotides,and a sense strand that consists of, consists essentially of, orcomprises a nucleotide sequence differing by no more than 1 nucleotidefrom the nucleotide sequence (5′→3′) GCCAAUAAAICUGGACAAGAA (SEQ IDNO:25), wherein all or substantially all of the nucleotides are modifiednucleotides.

In some embodiments, an APOC3 RNAi agent disclosed herein includes anantisense strand that consists of, consists essentially of, or comprisesa nucleobase sequence differing by 0 or 1 nucleobases from thenucleotide sequence (5′→3′) AGAAUACUGUCCCUUUUAGGG (SEQ ID NO:10) and asense strand that consists of, consists essentially of, or comprises anucleobase sequence differing by 0 or 1 nucleobases from the nucleotidesequence (5′→3′) CCCUAAAAGGGACAGUAUUCU (SEQ ID NO:27). In someembodiments, an APOC3 RNAi agent disclosed herein includes an antisensestrand that consists of, consists essentially of, or comprises anucleotide sequence differing by no more than 1 nucleotide from thenucleotide sequence (5′→3′) AGAAUACUGUCCCUUUUAGGG (SEQ ID NO:10),wherein all or substantially all of the nucleotides are modifiednucleotides, and a sense strand that consists of, consists essentiallyof, or comprises a nucleotide sequence differing by no more than 1nucleotide from the nucleotide sequence (5′→3′) CCCUAAAAGGGACAGUAUUCU(SEQ ID NO:27), wherein all or substantially all of the nucleotides aremodified nucleotides.

In some embodiments, an APOC3 RNAi agent disclosed herein includes anantisense strand that consists of, consists essentially of, or comprisesa nucleobase sequence differing by 0 or 1 nucleobases from thenucleotide sequence (5′→3′) AGAAUACUGUCCCUUUUAAGC (SEQ ID NO:12) and asense strand that consists of, consists essentially of, or comprises anucleobase sequence differing by 0 or 1 nucleobases from the nucleotidesequence (5′→3′) GCUUAAAAGGGACAGUAUUCU (SEQ ID NO:29). In someembodiments, an APOC3 RNAi agent disclosed herein includes an antisensestrand that consists of, consists essentially of, or comprises anucleotide sequence differing by no more than 1 nucleotide from thenucleotide sequence (5′→3′) AGAAUACUGUCCCUUUUAAGC (SEQ ID NO: 12),wherein all or substantially all of the nucleotides are modifiednucleotides, and a sense strand that consists of, consists essentiallyof, or comprises a nucleotide sequence differing by no more than 1nucleotide from the nucleotide sequence (5′→3′) GCUUAAAAGGGACAGUAUUCU(SEQ ID NO:29), wherein all or substantially all of the nucleotides aremodified nucleotides.

In some embodiments, an APOC3 RNAi agent disclosed herein includes anantisense strand that consists of, consists essentially of, or comprisesa nucleobase sequence differing by 0 or 1 nucleobases from thenucleotide sequence (5′→3′) UGAGAAUACUGUCCCUUUGCC (SEQ ID NO:14) and asense strand that consists of, consists essentially of, or comprises anucleobase sequence differing by 0 or 1 nucleobases from the nucleotidesequence (5′→3′) GGCAAAGGGACAGUAUUCUCA (SEQ ID NO:31). In someembodiments, an APOC3 RNAi agent disclosed herein includes an antisensestrand that consists of, consists essentially of, or comprises anucleotide sequence differing by no more than 1 nucleotide from thenucleotide sequence (5′→3′) UGAGAAUACUGUCCCUUUGCC (SEQ ID NO: 14),wherein all or substantially all of the nucleotides are modifiednucleotides, and a sense strand that consists of, consists essentiallyof, or comprises a nucleotide sequence differing by no more than 1nucleotide from the nucleotide sequence (5′→3′) GGCAAAGGGACAGUAUUCUCA(SEQ ID NO:31), wherein all or substantially all of the nucleotides aremodified nucleotides.

In some embodiments, an APOC3 RNAi agent disclosed herein includes anantisense strand that consists of, consists essentially of, or comprisesthe modified nucleotide sequence (5′-3′)usCfsasCfuGfagaauAfcUfgUfcCfcUfsc (SEQ ID NO:2), and a sense strand thatconsists of, consists essentially of, or comprises the modifiednucleotide sequence (5′→3′) gagggacaGfUfAfuucucaguia (SEQ ID NO: 15),wherein a, c, g, i, and u represent 2′-O-methyl adenosine, cytidine,guanosine, inosine, or uridine, respectively; Af, Cf, Gf, and Ufrepresent 2′-fluoro adenosine, cytidine, guanosine, or uridine,respectively; and s represents a phosphorothioate linkage. In someembodiments, an APOC3 RNAi agent disclosed herein includes an antisensestrand that consists of, consists essentially of, or comprises themodified nucleotide sequence (5′→3′) usCfsasCfuGfagaauAfcUfgUfcCfcUfsc(SEQ ID NO:2), and a sense strand that consists of, consists essentiallyof, or comprises the modified nucleotide sequence (5′→3′)gagggacaGfUfAfuucucaguia (SEQ ID NO: 15), and wherein the sense strandfurther includes inverted abasic residues at the 3′ terminal end and atthe 5′ end of the nucleotide sequence, and the sense strand alsoincludes a targeting ligand that is covalently linked to the 5′ terminalend, wherein the targeting ligand includes N-acetyl-galactosamine.

In some embodiments, an APOC3 RNAi agent disclosed herein includes anantisense strand that consists of, consists essentially of, or comprisesthe modified nucleotide sequence (5′-3′)usCfsasCfuGfagaauAfcUfgUfcCfcGfsu (SEQ ID NO:4), and a sense strand thatconsists of, consists essentially of, or comprises the modifiednucleotide sequence (5′→3′) acgggacaGfUfAfuucucaguia (SEQ ID NO: 17),wherein a, c, g, i, and u represent 2′-O-methyl adenosine, cytidine,guanosine, inosine, or uridine, respectively; Af, Cf, Gf, and Ufrepresent 2′-fluoro adenosine, cytidine, guanosine, or uridine,respectively; and s represents a phosphorothioate linkage. In someembodiments, an APOC3 RNAi agent disclosed herein includes an antisensestrand that consists of, consists essentially of, or comprises themodified nucleotide sequence (5′→3′) usCfsasCfuGfagaauAfcUfgUfcCfcGfsu(SEQ ID NO:4), and a sense strand that consists of, consists essentiallyof, or comprises the modified nucleotide sequence (5′→3′)acgggacaGfUfAfuucucaguia (SEQ ID NO: 17), and wherein the sense strandfurther includes inverted abasic residues at the 3′ terminal end and atthe 5′ end of the nucleotide sequence, and the sense strand alsoincludes a targeting ligand that is covalently linked to the 5′ terminalend, wherein the targeting ligand includes N-acetyl-galactosamine.

In some embodiments, an APOC3 RNAi agent disclosed herein includes anantisense strand that consists of, consists essentially of, or comprisesthe modified nucleotide sequence (5′-3′) usCfsascugagaauAfcUfgUfcCfcUfsc(SEQ ID NO:6), and a sense strand that consists of, consists essentiallyof, or comprises the modified nucleotide sequence (5′→3′)gagggacaGfuAfuUfcucaguia (SEQ ID NO: 19), wherein a, c, g, i, and urepresent 2′-O-methyl adenosine, cytidine, guanosine, inosine, oruridine, respectively; Af, Cf, Gf, and Uf represent 2′-fluoro adenosine,cytidine, guanosine, or uridine, respectively; and s represents aphosphorothioate linkage. In some embodiments, an APOC3 RNAi agentdisclosed herein includes an antisense strand that consists of, consistsessentially of, or comprises the modified nucleotide sequence (5′→3′)usCfsascugagaauAfcUfgUfcCfcUfsc (SEQ ID NO:6), and a sense strand thatconsists of, consists essentially of, or comprises the modifiednucleotide sequence (5′→3′) gagggacaGfuAfuUfcucaguia (SEQ ID NO: 19),and wherein the sense strand further includes inverted abasic residuesat the 3′ terminal end and at the 5′ end of the nucleotide sequence, andthe sense strand also includes a targeting ligand that is covalentlylinked to the 5′ terminal end, wherein the targeting ligand includesN-acetyl-galactosamine.

In some embodiments, an APOC3 RNAi agent disclosed herein includes anantisense strand that consists of, consists essentially of, or comprisesthe modified nucleotide sequence (5′-3′)usCfsasCfuGfagaauAfcUfgUfcCfcUfsc (SEQ ID NO:2), and a sense strand thatconsists of, consists essentially of, or comprises the modifiednucleotide sequence (5′→3′) gagggacaGfUfAfuucucaguga (SEQ ID NO:20),wherein a, c, g, and u represent 2′-O-methyl adenosine, cytidine,guanosine, or uridine, respectively; Af, Cf, Gf, and Uf represent2′-fluoro adenosine, cytidine, guanosine, or uridine, respectively; ands represents a phosphorothioate linkage. In some embodiments, an APOC3RNAi agent disclosed herein includes an antisense strand that consistsof, consists essentially of, or comprises the modified nucleotidesequence (5′→3′) usCfsasCfuGfagaauAfcUfgUfcCfcUfsc (SEQ ID NO:2), and asense strand that consists of, consists essentially of, or comprises themodified nucleotide sequence (5′→3′) gagggacaGfUfAfuucucaguga (SEQ IDNO:20), and wherein the sense strand further includes inverted abasicresidues at the 3′ terminal end and at the 5′ end of the nucleotidesequence, and the sense strand also includes a targeting ligand that iscovalently linked to the 5′ terminal end, wherein the targeting ligandincludes N-acetyl-galactosamine.

In some embodiments, an APOC3 RNAi agent disclosed herein includes anantisense strand that consists of, consists essentially of, or comprisesthe modified nucleotide sequence (5′-3′)usUfscsUfuGfuCfcAfgCfuUfuAfuUfgGfsc (SEQ ID NO:7), and a sense strandthat consists of, consists essentially of, or comprises the modifiednucleotide sequence (5′→3′) gccaauaaAfGfCfuggacaagaa (SEQ ID NO:22),wherein a, c, g, and u represent 2′-O-methyl adenosine, cytidine,guanosine, or uridine, respectively; Af, Cf, Gf, and Uf represent2′-fluoro adenosine, cytidine, guanosine, or uridine, respectively; ands represents a phosphorothioate linkage. In some embodiments, an APOC3RNAi agent disclosed herein includes an antisense strand that consistsof, consists essentially of, or comprises the modified nucleotidesequence (5′→3′) usUfscsUfuGfuCfcAfgCfuUfuAfuUfgGfsc (SEQ ID NO:7), anda sense strand that consists of, consists essentially of, or comprisesthe modified nucleotide sequence (5′→3′) gccaauaaAfGfCfuggacaagaa (SEQID NO:22), and wherein the sense strand further includes inverted abasicresidues at the 3′ terminal end and at the 5′ end of the nucleotidesequence, and the sense strand also includes a targeting ligand that iscovalently linked to the 5′ terminal end, wherein the targeting ligandincludes N-acetyl-galactosamine.

In some embodiments, an APOC3 RNAi agent disclosed herein includes anantisense strand that consists of, consists essentially of, or comprisesthe modified nucleotide sequence (5′-3′)usUfscsUfuGfuCfcAfgCfuUfuAfuUfgGfsc (SEQ ID NO:7), and a sense strandthat consists of, consists essentially of, or comprises the modifiednucleotide sequence (5′→3′) gccaauaaAflfCfuggacaagaa (SEQ ID NO:24),wherein a, c, g, and u represent 2′-O-methyl adenosine, cytidine,guanosine, or uridine, respectively; Af, Cf, Gf, If, and Uf represent2′-fluoro adenosine, cytidine, guanosine, inosine, or uridine,respectively; and s represents a phosphorothioate linkage. In someembodiments, an APOC3 RNAi agent disclosed herein includes an antisensestrand that consists of, consists essentially of, or comprises themodified nucleotide sequence (5′→3′) usUfscsUfuGfuCfcAfgCfuUfuAfuUfgGfsc(SEQ ID NO:7), and a sense strand that consists of, consists essentiallyof, or comprises the modified nucleotide sequence (5′→3′)gccaauaaAflfCfuggacaagaa (SEQ ID NO:24), and wherein the sense strandfurther includes inverted abasic residues at the 3′ terminal end and atthe 5′ end of the nucleotide sequence, and the sense strand alsoincludes a targeting ligand that is covalently linked to the 5′ terminalend, wherein the targeting ligand includes N-acetyl-galactosamine.

In some embodiments, an APOC3 RNAi agent disclosed herein includes anantisense strand that consists of, consists essentially of, or comprisesthe modified nucleotide sequence (5′-3′)asGfsasAfuAfcUfgUfcCfcUfuUfuAfgGfsg (SEQ ID NO:9), and a sense strandthat consists of, consists essentially of, or comprises the modifiednucleotide sequence (5′→3′) cccuaaaaGfGfGfacaguauucu (SEQ ID NO:26),wherein a, c, g, and u represent 2′-O-methyl adenosine, cytidine,guanosine, or uridine, respectively; Af, Cf, Gf, and Uf represent2′-fluoro adenosine, cytidine, guanosine, or uridine, respectively; ands represents a phosphorothioate linkage. In some embodiments, an APOC3RNAi agent disclosed herein includes an antisense strand that consistsof, consists essentially of, or comprises the modified nucleotidesequence (5′→3′) asGfsasAfuAfcUfgUfcCfcUfuUfuAfgGfsg (SEQ ID NO:9), anda sense strand that consists of, consists essentially of, or comprisesthe modified nucleotide sequence (5′→3′) cccuaaaaGfGfGfacaguauucu (SEQID NO:26), and wherein the sense strand further includes inverted abasicresidues at the 3′ terminal end and at the 5′ end of the nucleotidesequence, and the sense strand also includes a targeting ligand that iscovalently linked to the 5′ terminal end, wherein the targeting ligandincludes N-acetyl-galactosamine.

In some embodiments, an APOC3 RNAi agent disclosed herein includes anantisense strand that consists of, consists essentially of, or comprisesthe modified nucleotide sequence (5′-3′)asGfsasAfuAfcUfgUfcCfcUfuUfuAfaGfsc (SEQ ID NO:11), and a sense strandthat consists of, consists essentially of, or comprises the modifiednucleotide sequence (5′→3′) gcuuaaaaGfGfGfacaguauucu (SEQ ID NO:28),wherein a, c, g, and u represent 2′-O-methyl adenosine, cytidine,guanosine, or uridine, respectively; Af, Cf, Gf, and Uf represent2′-fluoro adenosine, cytidine, guanosine, or uridine, respectively; ands represents a phosphorothioate linkage. In some embodiments, an APOC3RNAi agent disclosed herein includes an antisense strand that consistsof, consists essentially of, or comprises the modified nucleotidesequence (5′→3′) asGfsasAfuAfcUfgUfcCfcUfuUfuAfaGfsc (SEQ ID NO: 11),and a sense strand that consists of, consists essentially of, orcomprises the modified nucleotide sequence (5′→3′)gcuuaaaaGfGfGfacaguauucu (SEQ ID NO:28), and wherein the sense strandfurther includes inverted abasic residues at the 3′ terminal end and atthe 5′ end of the nucleotide sequence, and the sense strand alsoincludes a targeting ligand that is covalently linked to the 5′ terminalend, wherein the targeting ligand includes N-acetyl-galactosamine.

In some embodiments, an APOC3 RNAi agent disclosed herein includes anantisense strand that consists of, consists essentially of, or comprisesthe modified nucleotide sequence (5′-3′)usGfsasGfaAfuAfcUfgUfcCfcUfuUfgcsc (SEQ ID NO:13), and a sense strandthat consists of, consists essentially of, or comprises the modifiednucleotide sequence (5′→3′) ggcaaaggGfAfCfaguauucuca (SEQ ID NO:30),wherein a, c, g, and u represent 2′-O-methyl adenosine, cytidine,guanosine, or uridine, respectively; Af, Cf, Gf, and Uf represent2′-fluoro adenosine, cytidine, guanosine, or uridine, respectively; ands represents a phosphorothioate linkage. In some embodiments, an APOC3RNAi agent disclosed herein includes an antisense strand that consistsof, consists essentially of, or comprises the modified nucleotidesequence (5′→3′) usGfsasGfaAfuAfcUfgUfcCfcUfuUfgcsc (SEQ ID NO:13), anda sense strand that consists of, consists essentially of, or comprisesthe modified nucleotide sequence (5′→3′) ggcaaaggGfAfCfaguauucuca (SEQID NO:30), and wherein the sense strand further includes inverted abasicresidues at the 3′ terminal end and at the 5′ end of the nucleotidesequence, and the sense strand also includes a targeting ligand that iscovalently linked to the 5′ terminal end, wherein the targeting ligandincludes N-acetyl-galactosamine.

In some embodiments, an APOC3 RNAi agent disclosed herein includes anantisense strand that consists of, consists essentially of, or comprisesa nucleotide sequence that differs by 0 or 1 nucleotides from one of thefollowing nucleotide sequences (5′→3′):

(SEQ ID NO: 3) UCACUGAGAAUACUGUCCCUC; (SEQ ID NO: 5)UCACUGAGAAUACUGUCCCGU; (SEQ ID NO: 8) UUCUUGUCCAGCUUUAUUGGC; (SEQ ID NO:10) AGAAUACUGUCCCUUUUAGGG; (SEQ ID NO: 12) AGAAUACUGUCCCUUUUAAGC; or(SEQ ID NO: 14) UGAGAAUACUGUCCCUUUGCC;

wherein the APOC3 RNAi agent further includes a sense strand that is atleast partially complementary to the antisense strand; and wherein allor substantially all of the nucleotides on both the antisense strand andthe sense strand are modified nucleotides.

In some embodiments, an APOC3 RNAi agent disclosed herein includes anantisense strand that consists of, consists essentially of, or comprisesa nucleotide sequence that differs by 0 or 1 nucleotides from one of thefollowing nucleotide sequences (5′→3′):

(SEQ ID NO: 3) UCACUGAGAAUACUGUCCCUC; (SEQ ID NO: 5)UCACUGAGAAUACUGUCCCGU; (SEQ ID NO: 8) UUCUUGUCCAGCUUUAUUGGC; (SEQ ID NO:10) AGAAUACUGUCCCUUUUAGGG; (SEQ ID NO: 12) AGAAUACUGUCCCUUUUAAGC; or(SEQ ID NO: 14) UGAGAAUACUGUCCCUUUGCC;

wherein the APOC3 RNAi agent further includes a sense strand that is atleast partially complementary to the antisense strand; wherein all orsubstantially all of the nucleotides on both the antisense strand andthe sense strand are modified nucleotides; and wherein the sense strandfurther includes inverted abasic residues at the 3′ terminal end and atthe 5′ end of the nucleotide sequence, and the sense strand alsoincludes a targeting ligand that is covalently linked to the 5′ terminalend, wherein the targeting ligand includes N-acetyl-galactosamine.

In some embodiments, an APOC3 RNAi agent disclosed herein includes anantisense strand that consists of, consists essentially of, or comprisesa nucleotide sequence that differs by 0 or 1 nucleotides from one of thefollowing nucleotide sequences (5′→3′):

(SEQ ID NO: 3) UCACUGAGAAUACUGUCCCUC; (SEQ ID NO: 5)UCACUGAGAAUACUGUCCCGU; (SEQ ID NO: 8) UUCUUGUCCAGCUUUAUUGGC; (SEQ ID NO:10) AGAAUACUGUCCCUUUUAGGG; (SEQ ID NO: 12) AGAAUACUGUCCCUUUUAAGC; or(SEQ ID NO: 14) UGAGAAUACUGUCCCUUUGCC;

wherein the APOC3 RNAi agent further includes a sense strand that is atleast partially complementary to the antisense strand; wherein all orsubstantially all of the nucleotides on both the antisense strand andthe sense strand are modified nucleotides; and wherein the sense strandfurther includes inverted abasic residues at the 3′ terminal end and atthe 5′ end of the nucleotide sequence, and the sense strand alsoincludes a targeting ligand that is covalently linked to the 5′ terminalend, wherein the targeting ligand includes N-acetyl-galactosamine; andwherein the respective antisense strand sequence is located at positions1-21 of the antisense strand.

In some embodiments, an APOC3 RNAi agent disclosed herein includes anantisense strand and a sense strand, wherein the antisense strand andthe sense strand consist of, consist essentially of, or comprisenucleotide sequences that differ by 0 or 1 nucleotides from one of thefollowing nucleotide sequence (5′→3′) pairs:

(SEQ ID NO: 3) UCACUGAGAAUACUGUCCCUC and (SEQ ID NO: 16)GAGGGACAGUAUUCUCAGUIA; (SEQ ID NO: 5) UCACUGAGAAUACUGUCCCGU and (SEQ IDNO: 18) ACGGGACAGUAUUCUCAGUIA; (SEQ ID NO: 3) UCACUGAGAAUACUGUCCCUC and(SEQ ID NO: 21) GAGGGACAGUAUUCUCAGUGA; (SEQ ID NO: 8)UUCUUGUCCAGCUUUAUUGGC and (SEQ ID NO: 23) GCCAAUAAAGCUGGACAAGAA; (SEQ IDNO: 8) UUCUUGUCCAGCUUUAUUGGC and (SEQ ID NO: 25) GCCAAUAAAICUGGACAAGAA;(SEQ ID NO: 10) AGAAUACUGUCCCUUUUAGGG and (SEQ ID NO: 27)CCCUAAAAGGGACAGUAUUCU; (SEQ ID NO: 12) AGAAUACUGUCCCUUUUAAGC and (SEQ IDNO: 29) GCUUAAAAGGGACAGUAUUCU; or (SEQ ID NO: 14) UGAGAAUACUGUCCCUUUGCCand (SEQ ID NO: 31) GGCAAAGGGACAGUAUUCUCA;

-   -   wherein all or substantially all of the nucleotides on both the        antisense strand and the sense strand are modified nucleotides.

In some embodiments, an APOC3 RNAi agent disclosed herein includes anantisense strand and a sense strand, wherein the antisense strand andthe sense strand consist of, consist essentially of, or comprisenucleotide sequences that differ by 0 or 1 nucleotides from one of thefollowing nucleotide sequences (5′→3′) pairs:

(SEQ ID NO: 3) UCACUGAGAAUACUGUCCCUC and (SEQ ID NO: 16)GAGGGACAGUAUUCUCAGUIA; (SEQ ID NO: 5) UCACUGAGAAUACUGUCCCGU and (SEQ IDNO: 18) ACGGGACAGUAUUCUCAGUIA; (SEQ ID NO: 3) UCACUGAGAAUACUGUCCCUC and(SEQ ID NO: 21) GAGGGACAGUAUUCUCAGUGA; (SEQ ID NO: 8)UUCUUGUCCAGCUUUAUUGGC and (SEQ ID NO: 23) GCCAAUAAAGCUGGACAAGAA; (SEQ IDNO: 8) UUCUUGUCCAGCUUUAUUGGC and (SEQ ID NO: 25) GCCAAUAAAICUGGACAAGAA;(SEQ ID NO: 10) AGAAUACUGUCCCUUUUAGGG and (SEQ ID NO: 27)CCCUAAAAGGGACAGUAUUCU; (SEQ ID NO: 12) AGAAUACUGUCCCUUUUAAGC and (SEQ IDNO: 29) GCUUAAAAGGGACAGUAUUCU; or (SEQ ID NO: 14) UGAGAAUACUGUCCCUUUGCCand (SEQ ID NO: 31) GGCAAAGGGACAGUAUUCUCA;

-   -   wherein all or substantially all of the nucleotides on both the        antisense strand and the sense strand are modified nucleotides;        and wherein the sense strand further includes inverted abasic        residues at the 3′ terminal end and at the 5′ end of the        nucleotide sequence, and the sense strand also includes a        targeting ligand that is covalently linked to the 5′ terminal        end, wherein the targeting ligand includes        N-acetyl-galactosamine.

In some embodiments, an APOC3 RNAi agent disclosed herein includes anantisense strand that consists of, consists essentially of, or comprisesa modified nucleotide sequence that differs by 0 or 1 nucleotides fromone of the following nucleotide sequences (5′→3′):

(SEQ ID NO: 2) usCfsasCfuGfagaauAfcUfgUfcCfcUfsc; (SEQ ID NO: 4)usCfsasCfuGfagaauAfcUfgUfcCfcGfsu; (SEQ ID NO: 6)usCfsascugagaauAfcUfgUfcCfcUfsc; (SEQ ID NO: 7)usUfscsUfuGfuCfcAfgCfuUfuAfuUfgGfsc; (SEQ ID NO: 9)asGfsasAfuAfcUfgUfcCfcUfuUfuAfgGfsg; (SEQ ID NO: 11)asGfsasAfuAfcUfgUfcCfcUfuUfuAfaGfsc; or (SEQ ID NO: 13)usGfsasGfaAfuAfcUfgUfcCfcUfuUfgcsc;

wherein a, c, g, and u represent 2′-O-methyl adenosine, cytidine,guanosine, or uridine, respectively; Af, Cf, Gf, and Uf represent2′-fluoro adenosine, cytidine, guanosine, or uridine, respectively; srepresents a phosphorothioate linkage; and wherein the APOC3 RNAi agentfurther includes the sense strand that is at least partiallycomplementary to the antisense strand; and wherein all or substantiallyall of the nucleotides on the sense strand are modified nucleotides.

In some embodiments, an APOC3 RNAi agent disclosed herein includes anantisense strand that consists of, consists essentially of, or comprisesa modified nucleotide sequence that differs by 0 or 1 nucleotides fromone of the following nucleotide sequences (5′→3′):

(SEQ ID NO: 2) usCfsasCfuGfagaauAfcUfgUfcCfcUfsc; (SEQ ID NO: 4)usCfsasCfuGfagaauAfcUfgUfcCfcGfsu; (SEQ ID NO: 6)usCfsascugagaauAfcUfgUfcCfcUfsc; (SEQ ID NO: 7)usUfscsUfuGfuCfcAfgCfuUfuAfuUfgGfsc; (SEQ ID NO: 9)asGfsasAfuAfcUfgUfcCfcUfuUfuAfgGfsg; (SEQ ID NO: 11)asGfsasAfuAfcUfgUfcCfcUfuUfuAfaGfsc; or (SEQ ID NO: 13)usGfsasGfaAfuAfcUfgUfcCfcUfuUfgcsc;

-   -   wherein the APOC3 RNAi agent further includes the sense strand        that is at least partially complementary to the antisense        strand; wherein all or substantially all of the nucleotides on        the sense strand are modified nucleotides; wherein all or        substantially all of the nucleotides on both the antisense        strand and the sense strand are modified nucleotides; and        wherein the sense strand further includes inverted abasic        residues at the 3′ terminal end and at the 5′ end of the        nucleotide sequence, and the sense strand also includes a        targeting ligand that is covalently linked to the 5′ terminal        end, wherein the targeting ligand includes        N-acetyl-galactosamine.

In some embodiments, an APOC3 RNAi agent disclosed herein includes anantisense strand and a sense strand that consists of, consistsessentially of, or comprises modified nucleotide sequences that differsby 0 or 1 nucleotides from one of the following nucleotide sequencepairs (5′→3′):

(SEQ ID NO: 2) usCfsasCfuGfagaauAfcUfgUfcCfcUfsc and (SEQ ID NO: 15)gagggacaGfUfAfuucucaguia; (SEQ ID NO: 4)usCfsasCfuGfagaauAfcUfgUfcCfcGfsu and (SEQ ID NO: 17)acgggacaGfUfAfuucucaguia; (SEQ ID NO: 6) usCfsascugagaauAfcUfgUfcCfcUfscand (SEQ ID NO: 19) gagggacaGfuAfuUfcucaguia; (SEQ ID NO: 2)usCfsasCfuGfagaauAfcUfgUfcCfcUfsc and (SEQ ID NO: 20)gagggacaGfUfAfuucucaguga; (SEQ ID NO: 7)usUfscsUfuGfuCfcAfgCfuUfuAfuUfgGfsc and (SEQ ID NO: 22)gccaauaaAfGfCfuggacaagaa; (SEQ ID NO: 7)usUfscsUfuGfuCfcAfgCfuUfuAfuUfgGfsc and (SEQ ID NO: 24)gccaauaaAfIfCfuggacaagaa; (SEQ ID NO: 9)asGfsasAfuAfcUfgUfcCfcUfuUfuAfgGfsg and (SEQ ID NO: 26)cccuaaaaGfGfGfacaguauucu; (SEQ ID NO: 11)asGfsasAfuAfcUfgUfcCfcUfuUfuAfaGfsc and (SEQ ID NO: 28)gcuuaaaaGfGfGfacaguauucu; or (SEQ ID NO: 13)usGfsasGfaAfuAfcUfgUfcCfcUfuUfgcsc and (SEQ ID NO: 30)ggcaaaggGfAfCfaguauucuca;

wherein a, c, g, i, and u represent 2′-O-methyl adenosine, cytidine,guanosine, inosine, or uridine, respectively; Af, Cf, Gf, If, and Ufrepresent 2′-fluoro adenosine, cytidine, guanosine, inosine or uridine,respectively; and s represents a phosphorothioate linkage.

In some embodiments, an APOC3 RNAi agent disclosed herein includes anantisense strand and a sense strand that consists of, consistsessentially of, or comprises one of the following nucleotide sequencepairs (5′→3′):

(SEQ ID NO: 2) usCfsasCfuGfagaauAfcUfgUfcCfcUfsc and (SEQ ID NO: 15)gagggacaGfUfAfuucucaguia; (SEQ ID NO: 4)usCfsasCfuGfagaauAfcUfgUfcCfcGfsu and (SEQ ID NO: 17)acgggacaGfUfAfuucucaguia; (SEQ ID NO: 6) usCfsascugagaauAfcUfgUfcCfcUfscand (SEQ ID NO: 19) gagggacaGfuAfuUfcucaguia; (SEQ ID NO: 2usCfsasCfuGfagaauAfcUfgUfcCfcUfsc and (SEQ ID NO: 20)gagggacaGfUfAfuucucaguga; (SEQ ID NO: 7)usUfscsUfuGfuCfcAfgCfuUfuAfuUfgGfsc and (SEQ ID NO: 22)gccaauaaAfGfCfuggacaagaa; (SEQ ID NO: 7)usUfscsUfuGfuCfcAfgCfuUfuAfuUfgGfsc and (SEQ ID NO: 24)gccaauaaAfIfCfuggacaagaa; (SEQ ID NO: 9)asGfsasAfuAfcUfgUfcCfcUfuUfuAfgGfsg and (SEQ ID NO: 26)cccuaaaaGfGfGfacaguauucu; (SEQ ID NO: 11)asGfsasAfuAfcUfgUfcCfcUfuUfuAfaGfsc and (SEQ ID NO: 28)gcuuaaaaGfGfGfacaguauucu; or (SEQ ID NO: 13)usGfsasGfaAfuAfcUfgUfcCfcUfuUfgcsc and (SEQ ID NO: 30)ggcaaaggGfAfCfaguauucuca;

wherein a, c, g, i, and u represent 2′-O-methyl adenosine, cytidine,guanosine, inosine, or uridine, respectively; Af, Cf, Gf, If, and Ufrepresent 2′-fluoro adenosine, cytidine, guanosine, inosine or uridine,respectively; s represents a phosphorothioate linkage; and wherein thesense strand further includes inverted abasic residues at the 3′terminal end and at the 5′ end of the nucleotide sequence, and the sensestrand also includes a targeting ligand that is covalently linked to the5′ terminal end, wherein the targeting ligand includesN-acetyl-galactosamine.

In some embodiments, an APOC3 RNAi agent disclosed herein includes anantisense strand that includes a nucleobase sequence that differs by 0or 1 nucleobases from the nucleotide sequences selected from the groupconsisting of (5′→3′):

(SEQ ID NO: 49) UCACUGAGAAUACUGUCCC; (SEQ ID NO: 53)UUCUUGUCCAGCUUUAUUG; (SEQ ID NO: 57) AGAAUACUGUCCCUUUUAA; (SEQ ID NO:58) AGAAUACUGUCCCUUUUAG; or (SEQ ID NO: 106) UGAGAAUACUGUCCCUUUG.

In some embodiments, an APOC3 RNAi agent disclosed herein includes anantisense strand that includes a nucleobase sequence that differs by 0or 1 nucleobases from the nucleotide sequences selected from the groupconsisting of (5′→3′):

(SEQ ID NO: 49) UCACUGAGAAUACUGUCCC; (SEQ ID NO: 53)UUCUUGUCCAGCUUUAUUG; (SEQ ID NO: 57) AGAAUACUGUCCCUUUUAA; (SEQ ID NO:58) AGAAUACUGUCCCUUUUAG; or (SEQ ID NO: 106) UGAGAAUACUGUCCCUUUG; and

wherein all or substantially all of the nucleotides are modifiednucleotides.

In some embodiments, an APOC3 RNAi agent disclosed herein includes anantisense strand that includes a nucleobase sequence that differs by 0or 1 nucleobases from the nucleotide sequences selected from the groupconsisting of (5′→3′):

(SEQ ID NO: 49) UCACUGAGAAUACUGUCCC; (SEQ ID NO: 53)UUCUUGUCCAGCUUUAUUG; (SEQ ID NO: 57) AGAAUACUGUCCCUUUUAA; (SEQ ID NO:58) AGAAUACUGUCCCUUUUAG; or (SEQ ID NO: 106) UGAGAAUACUGUCCCUUUG; and

wherein all or substantially all of the nucleotides are modifiednucleotides, and wherein SEQ ID NO:49, SEQ ID NO:53, SEQ ID NO:57, SEQID NO:58, or SEQ ID NO:106, respectively, is located at nucleotidepositions 1-19 (5′→3′) of the antisense strand.

In some embodiments, an APOC3 RNAi agent disclosed herein includes anantisense strand and a sense strand that each include a nucleobasesequences that differs by 0 or 1 nucleobases from the nucleotidesequence pairs selected from the group consisting of (5′→3′):

(SEQ ID NO: 49) UCACUGAGAAUACUGUCCC and (SEQ ID NO: 113)GGGACAGUAUUCUCAGUIA; (SEQ ID NO: 49) UCACUGAGAAUACUGUCCC and (SEQ ID NO:112) GGGACAGUAUUCUCAGUGA; (SEQ ID NO: 53) UUCUUGUCCAGCUUUAUUG and (SEQID NO: 117) CAAUAAAGCUGGACAAGAA; (SEQ ID NO: 53) UUCUUGUCCAGCUUUAUUG and(SEQ ID NO: 118) CAAUAAAICUGGACAAGAA; (SEQ ID NO: 57)AGAAUACUGUCCCUUUUAA and (SEQ ID NO: 122) UUAAAAGGGACAGUAUUCU; (SEQ IDNO: 58) AGAAUACUGUCCCUUUUAG and (SEQ ID NO: 123) CUAAAAGGGACAGUAUUCU; or(SEQ ID NO: 106) UGAGAAUACUGUCCCUUUG and (SEQ ID NO: 171)CAAAGGGACAGUAUUCUCA.

In some embodiments, an APOC3 RNAi agent disclosed herein includes anantisense strand and a sense strand that each include a nucleobasesequences that differs by 0 or 1 nucleobases from the nucleotidesequence pairs selected from the group consisting of (5′→3′):

(SEQ ID NO: 49) UCACUGAGAAUACUGUCCC and (SEQ ID NO: 113)GGGACAGUAUUCUCAGUIA; (SEQ ID NO: 49) UCACUGAGAAUACUGUCCC and(SEQ ID NO: 112) GGGACAGUAUUCUCAGUGA; (SEQ ID NO: 53)UUCUUGUCCAGCUUUAUUG and (SEQ ID NO: 117) CAAUAAAGCUGGACAAGAA;(SEQ ID NO: 53) UUCUUGUCCAGCUUUAUUG and (SEQ ID NO: 118)CAAUAAAICUGGACAAGAA; (SEQ ID NO: 57) AGAAUACUGUCCCUUUUAA and(SEQ ID NO: 122) UUAAAAGGGACAGUAUUCU; (SEQ ID NO: 58)AGAAUACUGUCCCUUUUAG and (SEQ ID NO: 123) CUAAAAGGGACAGUAUUCU; or(SEQ ID NO: 106) UGAGAAUACUGUCCCUUUG and (SEQ ID NO: 171)CAAAGGGACAGUAUUCUCA;and

wherein all or substantially all of the nucleotides are modifiednucleotides.

As used herein, the terms “oligonucleotide” and “polynucleotide” mean apolymer of linked nucleosides each of which can be independentlymodified or unmodified.

As used herein, an “RNAi agent” (also referred to as an “RNAi trigger”)means a composition that contains an RNA or RNA-like (e.g., chemicallymodified RNA) oligonucleotide molecule that is capable of degrading orinhibiting (e.g., degrades or inhibits under appropriate conditions)translation of messenger RNA (mRNA) transcripts of a target mRNA in asequence specific manner. As used herein, RNAi agents may operatethrough the RNA interference mechanism (i.e., inducing RNA interferencethrough interaction with the RNA interference pathway machinery(RNA-induced silencing complex or RISC) of mammalian cells), or by anyalternative mechanism(s) or pathway(s). While it is believed that RNAiagents, as that term is used herein, operate primarily through the RNAinterference mechanism, the disclosed RNAi agents are not bound by orlimited to any particular pathway or mechanism of action. RNAi agentsdisclosed herein are comprised of a sense strand and an antisensestrand, and include, but are not limited to: short (or small)interfering RNAs (siRNAs), double stranded RNAs (dsRNA), micro RNAs(miRNAs), short hairpin RNAs (shRNA), and dicer substrates. Theantisense strand of the RNAi agents described herein is at leastpartially complementary to the mRNA being targeted (i.e., APOC3 mRNA).RNAi agents can include one or more modified nucleotides and/or one ormore non-phosphodiester linkages.

As used herein, the terms “silence,” “reduce,” “inhibit,”“down-regulate,” or “knockdown” when referring to expression of a givengene, mean that the expression of the gene, as measured by the level ofRNA transcribed from the gene or the level of polypeptide, protein, orprotein subunit translated from the mRNA in a cell, group of cells,tissue, organ, or subject in which the gene is transcribed, is reducedwhen the cell, group of cells, tissue, organ, or subject is treated withthe RNAi agents described herein as compared to a second cell, group ofcells, tissue, organ, or subject that has not or have not been sotreated.

As used herein, the terms “sequence” and “nucleotide sequence” mean asuccession or order of nucleobases or nucleotides, described with asuccession of letters using standard nomenclature.

As used herein, a “base,” “nucleotide base,” or “nucleobase,” is aheterocyclic pyrimidine or purine compound that is a component of anucleotide, and includes the primary purine bases adenine and guanine,and the primary pyrimidine bases cytosine, thymine, and uracil. Anucleobase may further be modified to include, without limitation,universal bases, hydrophobic bases, promiscuous bases, size-expandedbases, and fluorinated bases. (See, e.g., Modified Nucleosides inBiochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH,2008). The synthesis of such modified nucleobases (includingphosphoramidite compounds that include modified nucleobases) is known inthe art.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleobase or nucleotidesequence (e.g., RNAi agent sense strand or targeted mRNA) in relation toa second nucleobase or nucleotide sequence (e.g., RNAi agent antisensestrand or a single-stranded antisense oligonucleotide), means theability of an oligonucleotide or polynucleotide including the firstnucleotide sequence to hybridize (form base pair hydrogen bonds undermammalian physiological conditions (or similar conditions in vitro)) andform a duplex or double helical structure under certain standardconditions with an oligonucleotide or polynucleotide including thesecond nucleotide sequence. Complementary sequences include Watson-Crickbase pairs or non-Watson-Crick base pairs and include natural ormodified nucleotides or nucleotide mimics, at least to the extent thatthe above hybridization requirements are fulfilled. Sequence identity orcomplementarity is independent of modification. For example, a and Af,as defined herein, are complementary to U (or T) and identical to A forthe purposes of determining identity or complementarity.

As used herein, “perfectly complementary” or “fully complementary” meansthat in a hybridized pair of nucleobase or nucleotide sequencemolecules, all (100%) of the bases in a contiguous sequence of a firstoligonucleotide will hybridize with the same number of bases in acontiguous sequence of a second oligonucleotide. The contiguous sequencemay comprise all or a part of a first or second nucleotide sequence.

As used herein, “partially complementary” means that in a hybridizedpair of nucleobase or nucleotide sequence molecules, at least 70%, butnot all, of the bases in a contiguous sequence of a firstoligonucleotide will hybridize with the same number of bases in acontiguous sequence of a second oligonucleotide. The contiguous sequencemay comprise all or a part of a first or second nucleotide sequence.

As used herein, “substantially complementary” means that in a hybridizedpair of nucleobase or nucleotide sequence molecules, at least 85%, butnot all, of the bases in a contiguous sequence of a firstoligonucleotide will hybridize with the same number of bases in acontiguous sequence of a second oligonucleotide. The contiguous sequencemay comprise all or a part of a first or second nucleotide sequence.

As used herein, the terms “complementary,” “fully complementary,”“partially complementary,” and “substantially complementary” are usedwith respect to the nucleobase or nucleotide matching between the sensestrand and the antisense strand of an RNAi agent, or between theantisense strand of an RNAi agent and a sequence of an APOC3 mRNA.

As used herein, the term “substantially identical” or “substantialidentity,” as applied to a nucleic acid sequence means the nucleotidesequence (or a portion of a nucleotide sequence) has at least about 85%sequence identity or more, e.g., at least 90%, at least 95%, or at least99% identity, compared to a reference sequence. Percentage of sequenceidentity is determined by comparing two optimally aligned sequences overa comparison window. The percentage is calculated by determining thenumber of positions at which the same type of nucleic acid base occursin both sequences to yield the number of matched positions, dividing thenumber of matched positions by the total number of positions in thewindow of comparison and multiplying the result by 100 to yield thepercentage of sequence identity. The inventions disclosed hereinencompass nucleotide sequences substantially identical to thosedisclosed herein.

As used herein, the terms “treat,” “treatment,” and the like, mean themethods or steps taken to provide relief from or alleviation of thenumber, severity, and/or frequency of one or more symptoms of a diseasein a subject. As used herein, “treat” and “treatment” may include thepreventative treatment, management, prophylactic treatment, and/orinhibition or reduction of the number, severity, and/or frequency of oneor more symptoms of a disease in a subject.

As used herein, the phrase “introducing into a cell,” when referring toan RNAi agent, means functionally delivering the RNAi agent into a cell.The phrase “functional delivery,” means delivering the RNAi agent to thecell in a manner that enables the RNAi agent to have the expectedbiological activity, e.g., sequence-specific inhibition of geneexpression.

Unless stated otherwise, use of the symbol

as used herein means that any group or groups may be linked thereto thatis in accordance with the scope of the inventions described herein.

As used herein, the term “isomers” refers to compounds that haveidentical molecular formulae, but that differ in the nature or thesequence of bonding of their atoms or in the arrangement of their atomsin space. Isomers that differ in the arrangement of their atoms in spaceare termed “stereoisomers.” Stereoisomers that are not mirror images ofone another are termed “diastereoisomers,” and stereoisomers that arenon-superimposable mirror images are termed “enantiomers,” or sometimesoptical isomers. A carbon atom bonded to four non-identical substituentsis termed a “chiral center.”

As used herein, unless specifically identified in a structure as havinga particular conformation, for each structure in which asymmetriccenters are present and thus give rise to enantiomers, diastereomers, orother stereoisomeric configurations, each structure disclosed herein isintended to represent all such possible isomers, including theiroptically pure and racemic forms. For example, the structures disclosedherein are intended to cover mixtures of diastereomers as well as singlestereoisomers.

As used in a claim herein, the phrase “consisting of” excludes anyelement, step, or ingredient not specified in the claim. When used in aclaim herein, the phrase “consisting essentially of” limits the scope ofa claim to the specified materials or steps and those that do notmaterially affect the basic and novel characteristic(s) of the claimedinvention.

The person of ordinary skill in the art would readily understand andappreciate that the compounds and compositions disclosed herein may havecertain atoms (e.g., N, O, or S atoms) in a protonated or deprotonatedstate, depending upon the environment in which the compound orcomposition is placed. Accordingly, as used herein, the structuresdisclosed herein envisage that certain functional groups, such as, forexample, OH, SH, or NH, may be protonated or deprotonated. Thedisclosure herein is intended to cover the disclosed compounds andcompositions regardless of their state of protonation based on theenvironment (such as pH), as would be readily understood by the personof ordinary skill in the art.

As used herein, the term “linked” or “conjugated” when referring to theconnection between two compounds or molecules means that two compoundsor molecules are joined by a covalent bond. Unless stated, the terms“linked” and “conjugated” as used herein may refer to the connectionbetween a first compound and a second compound either with or withoutany intervening atoms or groups of atoms.

As used herein, the term “including” is used to herein mean, and is usedinterchangeably with, the phrase “including but not limited to.” Theterm “or” is used herein to mean, and is used interchangeably with, theterm “and/or,” unless the context clearly indicates otherwise.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

Other objects, features, aspects, and advantages of the invention willbe apparent from the following detailed description, accompanyingfigures, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A. Schematic diagram of the modified sense and antisense strandsof APOC3 RNAi agent AD05251 (see Tables 4-6), conjugated to a tridentateN-acetyl-galactosamine-containing targeting ligand having the structureof (NAG37)s (see Table 7). FIG. 1A discloses SEQ ID NOs: 2 and 501.

The following abbreviations are used in FIGS. 1A to 11I: a, c, g, i, andu are 2′-O-methyl modified nucleotides (for i, the nucleobase ishypoxanthine (i.e., the base for inosine nucleotides)); Af, Cf, Gf, If,and Uf are 2′-fluoro modified nucleotides (for I, the nucleobase ishypoxanthine (i.e., the base for inosine nucleotides); p is aphosphodiester linkage; s is a phosphorothioate linkage; invAb is aninverted abasic (deoxyribose) residue (see Table 7); and (NAG37)s is atridentate N-acetyl-galactosamine targeting ligand having the structuredepicted in Table 7.

FIG. 1B. Schematic diagram of the modified sense and antisense strandsof APOC3 RNAi agent AD05876 (see Tables 4-6), conjugated to a tridentateN-acetyl-galactosamine-containing targeting ligand having the structureof (NAG37)s (see Table 7). FIG. 1B discloses SEQ ID NOs: 4 and 572.

FIG. 1C. Schematic diagram of the modified sense and antisense strandsof APOC3 RNAi agent AD05769 (see Tables 4-6), conjugated to a tridentateN-acetyl-galactosamine-containing targeting ligand having the structureof (NAG37)s (see Table 7). FIG. 1C discloses SEQ ID NOs: 6 and 557.

FIG. 1D. Schematic diagram of the modified sense and antisense strandsof APOC3 RNAi agent AD05169 (see Tables 4-6), conjugated to a tridentateN-acetyl-galactosamine-containing targeting ligand having the structureof (NAG37)s (see Table 7). FIG. 1D discloses SEQ ID NOs: 2 and 482.

FIG. 1E. Schematic diagram of the modified sense and antisense strandsof APOC3 RNAi agent AD05220 (see Tables 4-6), conjugated to a tridentateN-acetyl-galactosamine-containing targeting ligand having the structureof (NAG37)s (see Table 7). FIG. 1E discloses SEQ ID NOs: 7 and 494.

FIG. 1F. Schematic diagram of the modified sense and antisense strandsof APOC3 RNAi agent AD05547 (see Tables 4-6), conjugated to a tridentateN-acetyl-galactosamine-containing targeting ligand having the structureof (NAG37)s (see Table 7). FIG. 1F discloses SEQ ID NOs: 7 and 545.

FIG. 1G. Schematic diagram of the modified sense and antisense strandsof APOC3 RNAi agent AD05299 (see Tables 4-6), conjugated to a tridentateN-acetyl-galactosamine-containing targeting ligand having the structureof (NAG37)s (see Table 7). FIG. 1G discloses SEQ ID NOs: 9 and 521.

FIG. 1H. Schematic diagram of the modified sense and antisense strandsof APOC3 RNAi agent AD05223 (see Tables 4-6), conjugated to a tridentateN-acetyl-galactosamine-containing targeting ligand having the structureof (NAG37)s (see Table 7). FIG. 1H discloses SEQ ID NOs: 11 and 497.

FIG. 1I. Schematic diagram of the modified sense and antisense strandsof APOC3 RNAi agent AD05171 (see Tables 4-6), conjugated to a tridentateN-acetyl-galactosamine-containing targeting ligand having the structureof (NAG37)s (see Table 7). FIG. 1A discloses SEQ ID NOs: 13 and 483.

FIG. 2A to 2D. Chemical structure representation of APOC3 RNAi agentAD05251, including a tridentate N-acetyl-galactosamine-containingtargeting ligand (having the structure of (NAG37)s) conjugated at the 5′terminal end of the sense strand, shown as a free acid.

FIG. 3A to 3D. Chemical structure representation of APOC3 RNAi agentAD05251, including a tridentate N-acetyl-galactosamine-containingtargeting ligand (having the structure of (NAG37)s) conjugated at the 5′terminal end of the sense strand, shown as a sodium salt.

FIG. 4A to 4D. Chemical structure representation of APOC3 RNAi agentAD05876, including a tridentate N-acetyl-galactosamine-containingtargeting ligand (having the structure of (NAG37)s) conjugated at the 5′terminal end of the sense strand, shown as a free acid.

FIG. 5A to 5D. Chemical structure representation of APOC3 RNAi agentAD05876, including a tridentate N-acetyl-galactosamine-containingtargeting ligand (having the structure of (NAG37)s) conjugated at the 5′terminal end of the sense strand, shown as a sodium salt.

FIG. 6A to 6D. Chemical structure representation of APOC3 RNAi agentAD05220, including a tridentate N-acetyl-galactosamine-containingtargeting ligand (having the structure of (NAG37)s) conjugated at the 5′terminal end of the sense strand, shown as a free acid.

FIG. 7A to 7D. Chemical structure representation of APOC3 RNAi agentAD05220, including a tridentate N-acetyl-galactosamine-containingtargeting ligand (having the structure of (NAG37)s) conjugated at the 5′terminal end of the sense strand, shown as a sodium salt.

DETAILED DESCRIPTION

RNAi Agents

RNAi agents for inhibiting expression of an APOC3 gene (referred toherein as APOC3 RNAi agents or APOC3 RNAi triggers) are describedherein. Each APOC3 RNAi agent comprises a sense strand and an antisensestrand. The sense strand and the antisense strand each can be 16 to 30nucleotides in length. The sense and antisense strands can be either thesame length or they can be different lengths. In some embodiments, thesense and antisense strands are each independently 17 to 27 nucleotidesin length. In some embodiments, the sense and antisense strands are eachindependently 17-21 nucleotides in length. In some embodiments, thesense and antisense strands are each 21-26 nucleotides in length. Insome embodiments, the sense and antisense strands are each 21-24nucleotides in length. In some embodiments, the sense strand is about 19nucleotides in length while the antisense strand is about 21 nucleotidesin length. In some embodiments, the sense strand is about 21 nucleotidesin length while the antisense strand is about 23 nucleotides in length.In some embodiments, a sense strand is 23 nucleotides in length and anantisense strand is 21 nucleotides in length. In some embodiments, boththe sense and antisense strands are each 21 nucleotides in length. Insome embodiments, the RNAi agent sense and antisense strands are eachindependently 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides inlength. In some embodiments, a double-stranded RNAi agent has a duplexlength of about 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides.

In some embodiments, the region of perfect, substantial, or partialcomplementarity between the sense strand and the antisense strand is16-26 (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26) nucleotidesin length and occurs at or near the 5′ end of the antisense strand(e.g., this region may be separated from the 5′ end of the antisensestrand by 0, 1, 2, 3, or 4 nucleotides that are not perfectly,substantially, or partially complementary).

The sense strand and antisense strand each contain a core stretch (alsoreferred to herein as a “core sequence” or a “core stretch sequence”)that is 16 to 23 nucleotides in length. An antisense strand core stretchis 100% (perfectly) complementary or at least about 85% (substantially)complementary to a nucleotide sequence (sometimes referred to, e.g., asa target sequence) present in the APOC3 mRNA target. A sense strand corestretch sequence is 100% (perfectly) complementary or at least about 85%(substantially) complementary to a core stretch sequence in theantisense strand, and thus the sense strand core stretch sequence istypically perfectly identical or at least about 85% identical to anucleotide sequence (target sequence) present in the APOC3 mRNA target.A sense strand core stretch sequence can be the same length as acorresponding antisense core sequence or it can be a different length.In some embodiments, the antisense strand core stretch sequence is 16,17, 18, 19, 20, 21, 22, or 23 nucleotides in length. In someembodiments, the sense strand core stretch sequence is 16, 17, 18, 19,20, 21, 22, or 23 nucleotides in length.

Examples of sense and antisense strand nucleotide sequences used informing APOC3 RNAi agents are provided in Tables 2, 3, 4, and 5.Examples of RNAi agent duplexes, that include the sense strand andantisense strand sequences in Tables 2, 4, and 5, are shown in Table 6.

The APOC3 RNAi agent sense and antisense strands anneal to form aduplex. A sense strand and an antisense strand of an APOC3 RNAi agentcan be partially, substantially, or fully complementary to each other.Within the complementary duplex region, the sense strand core stretchsequence is at least 85% complementary or 100% complementary to theantisense core stretch sequence. In some embodiments, the sense strandcore stretch sequence contains a sequence of at least 16, at least 17,at least 18, at least 19, at least 20, at least 21, at least 22, or atleast 23 nucleotides that is at least 85% or 100% complementary to acorresponding 16, 17, 18, 19, 20, 21, 22, or 23 nucleotide sequence ofthe antisense strand core stretch sequence (i.e., the sense andantisense core stretch sequences of an APOC3 RNAi agent have a region ofat least 16, at least 17, at least 18, at least 19, at least 20, atleast 21, at least 22, or at least 23 nucleotides that is at least 85%base paired or 100% base paired.) In some embodiments, the antisensestrand of an APOC3 RNAi agent disclosed herein differs by 0, 1, 2, or 3nucleotides from any of the antisense strand sequences in Table 2, Table3, or Table 4. In some embodiments, the sense strand of an APOC3 RNAiagent disclosed herein differs by 0, 1, 2, or 3 nucleotides from any ofthe sense strand sequences in Table 2. Table 3, or Table 5.

The sense strand and/or the antisense strand can optionally andindependently contain an additional 1, 2, 3, 4, 5, or 6 nucleotides(extension) at the 3′ end, the 5′ end, or both the 3′ and 5′ ends of thecore stretch sequences. The antisense strand additional nucleotides, ifpresent, may or may not be complementary to the corresponding sequencein the APOC3 mRNA. The sense strand additional nucleotides, if present,may or may not be identical to the corresponding sequence in the APOC3mRNA. The antisense strand additional nucleotides, if present, may ormay not be complementary to the corresponding sense strand's additionalnucleotides, if present.

As used herein, an extension comprises 1, 2, 3, 4, 5, or 6 nucleotidesat the 5′ and/or 3′ end of the sense strand core stretch sequence and/orantisense strand core stretch sequence. The extension nucleotides on asense strand may or may not be complementary to nucleotides, either corestretch sequence nucleotides or extension nucleotides, in thecorresponding antisense strand. Conversely, the extension nucleotides onan antisense strand may or may not be complementary to nucleotides,either core stretch nucleotides or extension nucleotides, in thecorresponding sense strand. In some embodiments, both the sense strandand the antisense strand of an RNAi agent contain 3′ and 5′ extensions.In some embodiments, one or more of the 3′ extension nucleotides of onestrand base pairs with one or more 5′ extension nucleotides of the otherstrand. In other embodiments, one or more of 3′ extension nucleotides ofone strand do not base pair with one or more 5′ extension nucleotides ofthe other strand. In some embodiments, an APOC3 RNAi agent has anantisense strand having a 3′ extension and a sense strand having a 5′extension. In some embodiments, the extension nucleotide(s) are unpairedand form an overhang. As used herein, an “overhang” refers to a stretchof one or more unpaired nucleotides located at a terminal end of eitherthe sense strand or the antisense strand that does not form part of thehybridized or duplexed portion of an RNAi agent disclosed herein.

In some embodiments, an APOC3 RNAi agent comprises an antisense strandhaving a 3′ extension of 1, 2, 3, 4, 5, or 6 nucleotides in length. Inother embodiments, an APOC3 RNAi agent comprises an antisense strandhaving a 3′ extension of 1, 2, or 3 nucleotides in length. In someembodiments, one or more of the antisense strand extension nucleotidescomprise uracil or thymidine nucleotides or nucleotides that arecomplementary to the corresponding APOC3 mRNA sequence.

In some embodiments, the 3′ end of the antisense strand can includeabasic residues (Ab), which can also be referred to as an “abasic site”or “abasic nucleotide.” An abasic residue (Ab) is a nucleotide ornucleoside that lacks a nucleobase at the 1′ position of the sugarmoiety. (See, e.g., U.S. Pat. No. 5,998,203). In some embodiments, Ab orAbAb can be added to the 3′ end of the antisense strand.

In some embodiments, the sense strand or the antisense strand mayinclude a “terminal cap,” which as used herein is a non-nucleotidecompound or other moiety that can be incorporated at one or more terminiof a strand of an RNAi agent disclosed herein, and can provide the RNAiagent, in some instances, with certain beneficial properties, such as,for example, protection against exonuclease degradation. In someembodiments, inverted abasic residues (invAb) are added as terminal caps(see Table 76). (See, e.g., F. Czaudema, Nucleic Acids Res., 2003,31(11), 2705-16). Terminal caps are generally known in the art, andinclude, for example, inverted abasic residues as well as carbon chainssuch as a terminal C3, C6, or C12 groups. In some embodiments, aterminal cap is present at either the 5′ terminal end, the 3′ terminalend, or both the 5′ and 3′ terminal ends of the sense strand.

In some embodiments, an APOC3 RNAi agent comprises a sense strand havinga 3′ extension of 1, 2, 3, 4, or 5 nucleotides in length. In someembodiments, one or more of the sense strand extension nucleotidescomprises adenosine, uracil, or thymidine nucleotides, AT dinucleotide,or nucleotides that correspond to nucleotides in the APOC3 mRNAsequence. In some embodiments, the 3′ sense strand extension includes orconsists of one of the following sequences, but is not limited to: T,UT, TT, UU, UUT, TTT, or TTTT (each listed 5′ to 3′).

In some embodiments, the 3′ end of the sense strand may includeadditional abasic residues or inverted abasic terminal caps. In someembodiments, UUAb, UAb, or Ab are added to the 3′ end of the sensestrand.

In some embodiments, one or more inverted abasic residues (invAb) areadded to the 3′ end of the sense strand. In some embodiments, one ormore inverted abasic residues or inverted abasic sites are insertedbetween the targeting ligand and the nucleobase sequence of the sensestrand of the RNAi agent. In some embodiments, the inclusion of one ormore inverted abasic residues or inverted abasic sites at or near theterminal end or terminal ends of the sense strand of an RNAi agentallows for enhanced activity or other desired properties of an RNAiagent.

In some embodiments, an APOC3 RNAi agent comprises a sense strand havinga 5′ extension of 1, 2, 3, 4, 5, or 6 nucleotides in length. In someembodiments, one or more of the sense strand extension nucleotidescomprise uracil or adenosine nucleotides or nucleotides that correspondto nucleotides in the APOC3 mRNA sequence. In some embodiments, thesense strand 5′ extension is one of the following sequences, but is notlimited to: CA, AUAGGC, AUAGG, AUAG, AUA, A, AA, AC, GCA, GGCA, GGC,UAUCA, UAUC, UCA, UAU, U, UU (each listed 5′ to 3′). A sense strand canhave a 3′ extension and/or a 5′ extension.

In some embodiments, the 5′ end of the sense strand can include one ormore additional abasic residues (e.g., (Ab) or (AbAb)). In someembodiments, one or more inverted abasic residues (invAb) are added tothe 5′ end of the sense strand. In some embodiments, one or moreinverted abasic residues can be inserted between the targeting ligandand the nucleobase sequence of the sense strand of the RNAi agent. Insome embodiments, the inclusion of one or more inverted abasic residuesat or near the terminal end or terminal ends of the sense strand of anRNAi agent may allow for enhanced activity or other desired propertiesof an RNAi agent. In some embodiments, an abasic (deoxyribose) residuecan be replaced with a ribitol (abasic ribose) residue.

In some embodiments, the 3′ end of the antisense strand core stretchsequence, or the 3′ end of the antisense strand sequence, may include aninverted abasic residue (invAb (see Table 7)).

Examples of sequences used in forming APOC3 RNAi agents are provided inTables 2, 3, 4, and 5. In some embodiments, an APOC3 RNAi agentantisense strand includes a sequence of any of the sequences in Tables2, 3, or 4. In certain embodiments, an APOC3 RNAi agent antisense strandcomprises or consists of any one of the modified sequences in Table 4.In some embodiments, an APOC3 RNAi agent antisense strand includes thesequence of nucleotides (from 5′ end→3′ end) 1-17, 2-15, 2-17, 1-18,2-18, 1-19, 2-19, 1-20, 2-20, 1-21, 2-21, 1-22, 2-22, 1-23, 2-23, 1-24,or 2-24 of any of the sequences in Tables 2 or 4. In some embodiments,an APOC3 RNAi agent sense strand includes the sequence of any of thesequences in Tables 2 or 5. In some embodiments, an APOC3 RNAi agentsense strand includes the sequence of nucleotides (from 5′ end→3′ end)1-18, 1-19, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-26, 2-19, 2-20, 2-21,2-22, 2-23, 2-24, 3-20, 3-21, 3-22, 3-23, 3-24, 4-21, 4-22, 4-23, 4-24,5-22, 5-23, or 5-24 of any of the sequences in Tables 2 or 5. In certainembodiments, an APOC3 RNAi agent sense strand comprises or consists of amodified sequence of any one of the modified sequences in Table 5.

In some embodiments, the sense and antisense strands of the RNAi agentsdescribed herein contain the same number of nucleotides. In someembodiments, the sense and antisense strands of the RNAi agentsdescribed herein contain different numbers of nucleotides. In someembodiments, the sense strand 5′ end and the antisense strand 3′ end ofan RNAi agent form a blunt end. In some embodiments, the sense strand 3′end and the antisense strand 5′ end of an RNAi agent form a blunt end.In some embodiments, both ends of an RNAi agent form blunt ends. In someembodiments, neither end of an RNAi agent is blunt-ended. As used hereina “blunt end” refers to an end of a double stranded RNAi agent in whichthe terminal nucleotides of the two annealed strands are complementary(form a complementary base-pair).

In some embodiments, the sense strand 5′ end and the antisense strand 3′end of an RNAi agent form a frayed end. In some embodiments, the sensestrand 3′ end and the antisense strand 5′ end of an RNAi agent form afrayed end. In some embodiments, both ends of an RNAi agent form afrayed end. In some embodiments, neither end of an RNAi agent is afrayed end. As used herein a frayed end refers to an end of a doublestranded RNAi agent in which the terminal nucleotides of the twoannealed strands from a pair (i.e., do not form an overhang) but are notcomplementary (i.e. form a non-complementary pair). In some embodiments,one or more unpaired nucleotides at the end of one strand of a doublestranded RNAi agent form an overhang. The unpaired nucleotides may be onthe sense strand or the antisense strand, creating either 3′ or 5′overhangs. In some embodiments, the RNAi agent contains: a blunt end anda frayed end, a blunt end and 5′ overhang end, a blunt end and a 3′overhang end, a frayed end and a 5′ overhang end, a frayed end and a 3′overhang end, two 5′ overhang ends, two 3′ overhang ends, a 5′ overhangend and a 3′ overhang end, two frayed ends, or two blunt ends.

Typically, when present, overhangs are located at the 3′ terminal endsof the sense strand, the antisense strand, or both the sense strand andthe antisense strand.

Modified nucleotides, when used in various polynucleotide oroligonucleotide constructs, can preserve activity of the compound incells while at the same time increasing the serum stability of thesecompounds, and can also minimize the possibility of activatinginterferon activity in humans upon administering of the polynucleotideor oligonucleotide construct.

In some embodiments, an APOC3 RNAi agent is prepared or provided as asalt, mixed salt, or a free-acid. In some embodiments, an APOC3 RNAiagent is prepared as a sodium salt. Such forms that are well known inthe art are within the scope of the inventions disclosed herein.

Modified Nucleotides

In some embodiments, an APOC3 RNAi agent contains one or more modifiednucleotides. As used herein, a “modified nucleotide” is a nucleotideother than a ribonucleotide (2′-hydroxyl nucleotide). In someembodiments, at least 50% (e.g., at least 60%, at least 70%, at least80%, at least 90%, at least 95%, at least 97%, at least 98%, at least99%, or 100%) of the nucleotides are modified nucleotides. As usedherein, modified nucleotides can include, but are not limited to,deoxyribonucleotides, nucleotide mimics, abasic nucleotides (representedherein as Ab), 2′-modified nucleotides, 3′ to 3′ linkages (inverted)nucleotides (represented herein as invdN, invN, invn), modifiednucleobase-comprising nucleotides, bridged nucleotides, peptide nucleicacids (PNAs), 2′,3′-seco nucleotide mimics (unlocked nucleobaseanalogues, represented herein as N_(UNA or) NUNA), locked nucleotides(represented herein as N_(LNA) or NLNA), 3′-O-methoxy (2′internucleoside linked) nucleotides (represented herein as 3′-OMen),2′-F-Arabino nucleotides (represented herein as NfANA or Nf_(ANA)),5′-Me, 2′-fluoro nucleotide (represented herein as 5Me-Nf), morpholinonucleotides, vinyl phosphonate deoxyribonucleotides (represented hereinas vpdN), vinyl phosphonate containing nucleotides, and cyclopropylphosphonate containing nucleotides (cPrpN). 2′-modified nucleotides(i.e., a nucleotide with a group other than a hydroxyl group at the 2′position of the five-membered sugar ring) include, but are not limitedto, 2′-O-methyl nucleotides (represented herein as a lower case letter‘n’ in a nucleotide sequence), 2′-deoxy-2′-fluoro nucleotides (alsoreferred to herein as 2′-fluoro nucleotide, and represented herein asNf), 2′-deoxy nucleotides (represented herein as dN), 2′-methoxyethyl(2′-O-2-methoxylethyl) nucleotides (also referred to herein as 2′-MOE,and represented herein as NM), 2′-amino nucleotides, and 2′-alkylnucleotides. It is not necessary for all positions in a given compoundto be uniformly modified. Conversely, more than one modification can beincorporated in a single APOC3 RNAi agent or even in a single nucleotidethereof. The APOC3 RNAi agent sense strands and antisense strands can besynthesized and/or modified by methods known in the art. Modification atone nucleotide is independent of modification at another nucleotide.

Modified nucleobases include synthetic and natural nucleobases, such as5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6substituted purines, (e.g., 2-aminopropyladenine, 5-propynyluracil, or5-propynylcytosine), 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, inosine, xanthine, hypoxanthine, 2-aminoadenine, 6-alkyl(e.g., 6-methyl, 6-ethyl, 6-isopropyl, or 6-n-butyl) derivatives ofadenine and guanine, 2-alkyl (e.g., 2-methyl, 2-ethyl, 2-isopropyl, or2-n-butyl) and other alkyl derivatives of adenine and guanine,2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-halouracil, cytosine,5-propynyl uracil, 5-propynyl cytosine, 6-azo uracil, 6-azo cytosine,6-azo thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-sulfhydryl, 8-thioalkyl, 8-hydroxyl and other 8-substituted adeninesand guanines, 5-halo (e.g., 5-bromo), 5-trifluoromethyl, and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine,7-deazaadenine, 3-deazaguanine, and 3-deazaadenine.

In some embodiments, all or substantially all of the nucleotides of anRNAi agent are modified nucleotides. As used herein, an RNAi agentwherein substantially all of the nucleotides present are modifiednucleotides is an RNAi agent having four or fewer (i.e., 0, 1, 2, 3, or4) nucleotides in both the sense strand and the antisense strand beingribonucleotides (i.e., unmodified). As used herein, a sense strandwherein substantially all of the nucleotides present are modifiednucleotides is a sense strand having two or fewer (i.e., 0, 1, or 2)nucleotides in the sense strand being unmodified ribonucleotides. Asused herein, an antisense sense strand wherein substantially all of thenucleotides present are modified nucleotides is an antisense strandhaving two or fewer (i.e., 0, 1, or 2) nucleotides in the sense strandbeing unmodified ribonucleotides. In some embodiments, one or morenucleotides of an RNAi agent is an unmodified ribonucleotide.

Modified Internucleoside Linkages

In some embodiments, one or more nucleotides of an APOC3 RNAi agent arelinked by non-standard linkages or backbones (i.e., modifiedinternucleoside linkages or modified backbones). Modifiedinternucleoside linkages or backbones include, but are not limited to,phosphorothioate groups (represented herein as a lower case “s”), chiralphosphorothioates, thiophosphates, phosphorodithioates,phosphotriesters, aminoalkyl-phosphotriesters, alkyl phosphonates (e.g.,methyl phosphonates or 3′-alkylene phosphonates), chiral phosphonates,phosphinates, phosphoramidates (e.g., 3′-amino phosphoramidate,aminoalkylphosphoramidates, or thionophosphoramidates),thionoalkyl-phosphonates, thionoalkylphosphotriesters, morpholinolinkages, boranophosphates having normal 3′-5′ linkages, 2′-5′ linkedanalogs of boranophosphates, or boranophosphates having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′. In some embodiments, a modifiedinternucleoside linkage or backbone lacks a phosphorus atom. Modifiedinternucleoside linkages lacking a phosphorus atom include, but are notlimited to, short chain alkyl or cycloalkyl inter-sugar linkages, mixedheteroatom and alkyl or cycloalkyl inter-sugar linkages, or one or moreshort chain heteroatomic or heterocyclic inter-sugar linkages. In someembodiments, modified internucleoside backbones include, but are notlimited to, siloxane backbones, sulfide backbones, sulfoxide backbones,sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones,sulfamate backbones, methyleneimino and methylenehydrazino backbones,sulfonate and sulfonamide backbones, amide backbones, and otherbackbones having mixed N, O, S, and CH₂ components.

In some embodiments, a sense strand of an APOC3 RNAi agent can contain1, 2, 3, 4, 5, or 6 phosphorothioate linkages, an antisense strand of anAPOC3 RNAi agent can contain 1, 2, 3, 4, 5, or 6 phosphorothioatelinkages, or both the sense strand and the antisense strandindependently can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages.In some embodiments, a sense strand of an APOC3 RNAi agent can contain1, 2, 3, or 4 phosphorothioate linkages, an antisense strand of an APOC3RNAi agent can contain 1, 2, 3, or 4 phosphorothioate linkages, or boththe sense strand and the antisense strand independently can contain 1,2, 3, or 4 phosphorothioate linkages.

In some embodiments, an APOC3 RNAi agent sense strand contains at leasttwo phosphorothioate internucleoside linkages. In some embodiments, theat least two phosphorothioate internucleoside linkages are between thenucleotides at positions 1-3 from the 3′ end of the sense strand. Insome embodiments, one phosphorothioate internucleoside linkage is at the5′ end of the sense strand, and another phosphorothioate linkage is atthe 3′ end of the sense strand. In some embodiments, twophosphorothioate internucleoside linkage are located at the 5′ end ofthe sense strand, and another phosphorothioate linkage is at the 3′ endof the sense strand. In some embodiments, the sense strand dose notinclude any phosphorothioate internucleoside linkages between thenucleotides, but contains one, two, or three phosphorothioate linkagesbetween the terminal nucleotides on both the 5′ and 3′ ends and theoptionally present inverted abasic residue terminal caps. In someembodiments, the targeting ligand is linked to the sense strand via aphosphorothioate linkage.

In some embodiments, an APOC3 RNAi agent antisense strand contains fourphosphorothioate internucleoside linkages. In some embodiments, the fourphosphorothioate internucleoside linkages are between the nucleotides atpositions 1-3 from the 5′ end of the antisense strand and between thenucleotides at positions 19-21, 20-22, 21-23, 22-24, 23-25, or 24-26from the 5′ end. In some embodiments, three phosphorothioateinternucleoside linkages are located between positions 1-4 from the 5′end of the antisense strand, and a fourth phosphorothioateinternucleoside linkage is located between positions 20-21 from the 5′end of the antisense strand. In some embodiments, an APOC3 RNAi agentcontains at least three or four phosphorothioate internucleosidelinkages in the antisense strand.

In some embodiments, an APOC3 RNAi agent contains one or more modifiednucleotides and one or more modified internucleoside linkages. In someembodiments, a 2′-modified nucleoside is combined with modifiedinternucleoside linkage.

APOC3 RNAi Agents

In some embodiments, the APOC3 RNAi agents disclosed herein target anAPOC3 gene at or near the positions of the APOC3 gene show in Table 1.In some embodiments, the antisense strand of an APOC3 RNAi agentdisclosed herein includes a core stretch sequence that is fully,substantially, or at least partially complementary to a target APOC319-mer sequence disclosed in Table 1.

TABLE 1 APOC3 19-mer mRNA target sequences (taken fromhomo sapiens apolipoprotein C3 (APOC3)transcript, GenBank NM_000040.1 (SEQ ID NO: 1)). APOC3 19-merCorresponding SEQ Target Sequences Positions on ID No. (5′ → 3′)SEQ ID NO: 1 32 GGGACAGUAUUCUCAGUGC 438-456 33 CAAUAAAGCUGGACAAGAA506-524 34 UUAAAAGGGACAGUAUUCU 432-450 35 CGGGUACUCCUUGUUGUUG 56-74 36GGUACUCCUUGUUGUUGCC 58-76 37 GCUGGGUGACCGAUGGCUU 228-246 38GACCGAUGGCUUCAGUUCC 235-253 39 GCUUCAGUUCCCUGAAAGA 243-261 40UCAGUUCCCUGAAAGACUA 246-264 41 GACUACUGGAGCACCGUUA 260-278 42ACUACUGGAGCACCGUUAA 261-279 43 GCACCGUUAAGGACAAGUU 270-288 44ACCGUUAAGGACAAGUUCU 272-290 45 CCGUUAAGGACAAGUUCUC 273-291 46CCUCAAUACCCCAAGUCCA 349-367 47 AAAAGGGACAGUAUUCUCA 434-452 48AGGGACAGUAUUCUCAGUG 437-455

In some embodiments, an APOC3 RNAi agent includes an antisense strandwherein position 19 of the antisense strand (5′→3′) is capable offorming a base pair with position 1 of a 19-mer target sequencedisclosed in Table 1. In some embodiments, an APOC3 RNAi agent includesan antisense strand wherein position 1 of the antisense strand (5′→3′)is capable of forming a base pair with position 19 of the 19-mer targetsequence disclosed in Table 1.

In some embodiments, an APOC3 RNAi agent includes an antisense strandwherein position 2 of the antisense strand (5′→3′) is capable of forminga base pair with position 18 of the 19-mer target sequence disclosed inTable 1. In some embodiments, an APOC3 RNAi agent includes an antisensestrand wherein positions 2 through 18 of the antisense strand (5′→3′)are capable of forming base pairs with each of the respectivecomplementary bases located at positions 18 through 2 of the 19-mertarget sequence disclosed in Table 1.

For the RNAi agents disclosed herein, the nucleotide at position 1 ofthe antisense strand (from 5′ end→3′ end) can be perfectly complementaryto the APOC3 gene, or can be non-complementary to the APOC3 gene. Insome embodiments, the nucleotide at position 1 of the antisense strand(from 5′ end→3′ end) is a U, A, or dT. In some embodiments, thenucleotide at position 1 of the antisense strand (from 5′ end→3′ end)forms an A:U or U:A base pair with the sense strand.

In some embodiments, an APOC3 RNAi agent antisense strand comprises thesequence of nucleotides (from 5′ end→3′ end) 2-18 or 2-19 of any of theantisense strand sequences in Table 2, Table 3, or Table 4. In someembodiments, an APOC3 RNAi sense strand comprises the sequence ofnucleotides (from 5′ end→3′ end) 1-17, 1-18, or 2-18 of any of the sensestrand sequences in Table 2, Table 3, or Table 5.

In some embodiments, an APOC3 RNAi agent is comprised of (i) anantisense strand comprising the sequence of nucleotides (from 5′ end→3′end) 2-18 or 2-19 of any of the antisense strand sequences in Table 2,Table 3, or Table 4, and (ii) a sense strand comprising the sequence ofnucleotides (from 5′ end→3′ end) 1-17 or 1-18 of any of the sense strandsequences in Table 2, Table 3, or Table 5.

In some embodiments, the APOC3 RNAi agents include core 19-mernucleotide sequences shown in the following Table 2.

TABLE 2APOC3 RNAi Agent Antisense Strand and Sense Strand Core Stretch Base Sequences(N = any nucleobase). Antisense Strand Base Sense Strand Base SequenceSequence (5′ → 3′) (5′ → 3′) Corresponding SEQ (Shown as an Unmodified(Shown as an Unmodified Positions on ID No. Nucleotide Sequence)SEQ ID No. Nucleotide Sequence) SEQ ID NO: 1 49 UCACUGAGAAUACUGUCCC 114GGGACAGUAUUCUCAGUGA 438-456 49 UCACUGAGAAUACUGUCCC 115GGGACAGUAUUCUCAGUIA 438-456 50 GCACUGAGAAUACUGUCCC 116GGGACAGUAUUCUCAGUGC 438-456 51 NCACUGAGAAUACUGUCCC 117GGGACAGUAUUCUCAGUGN 438-456 52 NCACUGAGAAUACUGUCCN 118NGGACAGUAUUCUCAGUGN 438-456 53 UUCUUGUCCAGCUUUAUUG 119CAAUAAAGCUGGACAAGAA 506-524 53 UUCUUGUCCAGCUUUAUUG 120CAAUAAAICUGGACAAGAA 506-524 54 NUCUUGUCCAGCUUUAUUG 121CAAUAAAGCUGGACAAGAN 506-524 55 NUCUUGUCCAGCUUUAUUN 122NAAUAAAGCUGGACAAGAN 506-524 56 UGAAUACUGUCCCUUUUAA 123UUAAAAGGGACAGUAUUCA 432-450 57 AGAAUACUGUCCCUUUUAA 124UUAAAAGGGACAGUAUUCU 432-450 58 AGAAUACUGUCCCUUUUAG 125CUAAAAGGGACAGUAUUCU 432-450 59 NGAAUACUGUCCCUUUUAA 126UUAAAAGGGACAGUAUUCN 432-450 60 NGAAUACUGUCCCUUUUAG 127CUAAAAGGGACAGUAUUCN 432-450 61 NGAAUACUGUCCCUUUUAN 128NUAAAAGGGACAGUAUUCN 432-450 62 UAACAACAAGGAGUACCCG 129CGGGUACUCCUUGUUGUUA 56-74 63 CAACAACAAGGAGUACCCG 130 CGGGUACUCCUUGUUGUUG56-74 64 NAACAACAAGGAGUACCCG 131 CGGGUACUCCUUGUUGUUN 56-74 65NAACAACAAGGAGUACCCN 132 NGGGUACUCCUUGUUGUUN 56-74 66 UGCAACAACAAGGAGUACC133 GGUACUCCUUGUUGUUGCA 58-76 67 GGCAACAACAAGGAGUACC 134GGUACUCCUUGUUGUUGCC 58-76 68 NGCAACAACAAGGAGUACC 135 GGUACUCCUUGUUGUUGCN58-76 69 NGCAACAACAAGGAGUACN 136 NGUACUCCUUGUUGUUGCN 58-76 70UAGCCAUCGGUCACCCAGC 137 GCUGGGUGACCGAUGGCUA 228-246 71AAGCCAUCGGUCACCCAGC 138 GCUGGGUGACCGAUGGCUU 228-246 72NAGCCAUCGGUCACCCAGC 139 GCUGGGUGACCGAUGGCUN 228-246 73NAGCCAUCGGUCACCCAGN 140 NCUGGGUGACCGAUGGCUN 228-246 74UGAACUGAAGCCAUCGGUC 141 GACCGAUGGCUUCAGUUCA 235-253 75GGAACUGAAGCCAUCGGUC 142 GACCGAUGGCUUCAGUUCC 235-253 76NGAACUGAAGCCAUCGGUC 143 GACCGAUGGCUUCAGUUCN 235-253 77NGAACUGAAGCCAUCGGUN 144 NACCGAUGGCUUCAGUUCN 235-253 78UCUUUCAGGGAACUGAAGC 145 GCUUCAGUUCCCUGAAAGA 243-261 79NCUUUCAGGGAACUGAAGC 146 GCUUCAGUUCCCUGAAAGN 243-261 80NCUUUCAGGGAACUGAAGN 147 NCUUCAGUUCCCUGAAAGN 243-261 81UAGUCUUUCAGGGAACUGA 148 UCAGUUCCCUGAAAGACUA 246-264 82NAGUCUUUCAGGGAACUGA 149 UCAGUUCCCUGAAAGACUN 246-264 83NAGUCUUUCAGGGAACUGN 150 NCAGUUCCCUGAAAGACUN 246-264 84UAACGGUGCUCCAGUAGUC 151 GACUACUGGAGCACCGUUA 260-278 85NAACGGUGCUCCAGUAGUC 152 GACUACUGGAGCACCGUUN 260-278 86NAACGGUGCUCCAGUAGUN 153 NACUACUGGAGCACCGUUN 260-278 87UUAACGGUGCUCCAGUAGU 154 ACUACUGGAGCACCGUUAA 261-279 88NUAACGGUGCUCCAGUAGU 155 ACUACUGGAGCACCGUUAN 261-279 89NUAACGGUGCUCCAGUAGN 156 NCUACUGGAGCACCGUUAN 261-279 90UACUUGUCCUUAACGGUGC 157 GCACCGUUAAGGACAAGUA 270-288 91AACUUGUCCUUAACGGUGC 158 GCACCGUUAAGGACAAGUU 270-288 92NACUUGUCCUUAACGGUGC 159 GCACCGUUAAGGACAAGUN 270-288 93NACUUGUCCUUAACGGUGN 160 NCACCGUUAAGGACAAGUN 270-288 94UGAACUUGUCCUUAACGGU 161 ACCGUUAAGGACAAGUUCA 272-290 95AGAACUUGUCCUUAACGGU 162 ACCGUUAAGGACAAGUUCU 272-290 96NGAACUUGUCCUUAACGGU 163 ACCGUUAAGGACAAGUUCN 272-290 97NGAACUUGUCCUUAACGGN 164 NCCGUUAAGGACAAGUUCN 272-290 98UAGAACUUGUCCUUAACGG 165 CCGUUAAGGACAAGUUCUA 273-291 99GAGAACUUGUCCUUAACGG 166 CCGUUAAGGACAAGUUCUC 273-291 100NAGAACUUGUCCUUAACGG 167 CCGUUAAGGACAAGUUCUN 273-291 101NAGAACUUGUCCUUAACGN 168 NCGUUAAGGACAAGUUCUN 273-291 102UGGACUUGGGGUAUUGAGG 169 CCUCAAUACCCCAAGUCCA 349-367 103NGGACUUGGGGUAUUGAGG 170 CCUCAAUACCCCAAGUCCN 349-367 104NGGACUUGGGGUAUUGAGN 171 NCUCAAUACCCCAAGUCCN 349-367 105UGAGAAUACUGUCCCUUUU 172 AAAAGGGACAGUAUUCUCA 434-452 106UGAGAAUACUGUCCCUUUG 173 CAAAGGGACAGUAUUCUCA 434-452 107NGAGAAUACUGUCCCUUUU 174 AAAAGGGACAGUAUUCUCN 434-452 108NGAGAAUACUGUCCCUUUG 175 CAAAGGGACAGUAUUCUCN 434-452 109NGAGAAUACUGUCCCUUUN 176 NAAAGGGACAGUAUUCUCN 434-452 110UACUGAGAAUACUGUCCCU 177 AGGGACAGUAUUCUCAGUA 437-455 111CACUGAGAAUACUGUCCCU 178 AGGGACAGUAUUCUCAGUG 437-455 112NACUGAGAAUACUGUCCCU 179 AGGGACAGUAUUCUCAGUN 437-455 113NACUGAGAAUACUGUCCCN 180 NGGGACAGUAUUCUCAGUN 437-455

The APOC3 RNAi agent sense strands and antisense strands that compriseor consist of the nucleotide sequences in Table 2 can be modifiednucleotides or unmodified nucleotides. In some embodiments, the APOC3RNAi agents having the sense and antisense strand sequences thatcomprise or consist of the nucleotide sequences in Table 2 are all orsubstantially all modified nucleotides.

In some embodiments, the antisense strand of an APOC3 RNAi agentdisclosed herein differs by 0, 1, 2, or 3 nucleotides from any of theantisense strand sequences in Table 2. In some embodiments, the sensestrand of an APOC3 RNAi agent disclosed herein differs by 0, 1, 2, or 3nucleotides from any of the sense strand sequences in Table 2.

As used herein, each N listed in a sequence disclosed in Table 2 may beindependently selected from any and all nucleobases (including thosefound on both modified and unmodified nucleotides). In some embodiments,an N nucleotide listed in a sequence disclosed in Table 2 has anucleobase that is complementary to the N nucleotide at thecorresponding position on the other strand. In some embodiments, an Nnucleotide listed in a sequence disclosed in Table 2 has a nucleobasethat is not complementary to the N nucleotide at the correspondingposition on the other strand. In some embodiments, an N nucleotidelisted in a sequence disclosed in Table 2 has a nucleobase that is thesame as the N nucleotide at the corresponding position on the otherstrand. In some embodiments, an N nucleotide listed in a sequencedisclosed in Table 2 has a nucleobase that is different from the Nnucleotide at the corresponding position on the other strand.

Certain modified APOC3 RNAi agent antisense strands, as well as theirunderlying unmodified nucleobase sequences, are provided in Tables 3 and4. Certain modified APOC3 RNAi agent sense strands, as well as theirunderlying unmodified nucleobase sequences, are provided in Tables 3 and5. In forming APOC3 RNAi agents, each of the nucleotides in each of theunderlying base sequences listed in Tables 3, 4, and 5, as well as inTable 2. above, can be a modified nucleotide.

The APOC3 RNAi agents described herein are formed by annealing anantisense strand with a sense strand. A sense strand containing asequence listed in Table 2, Table 3, or Table 5, can be hybridized toany antisense strand containing a sequence listed in Table 2, Table 3,or Table 4, provided the two sequences have a region of at least 85%complementarity over a contiguous 16, 17, 18, 19, 20, or 21 nucleotidesequence.

In some embodiments, an APOC3 RNAi agent antisense strand comprises anucleotide sequence of any of the sequences in Table 2, Table 3, orTable 4.

In some embodiments, an APOC3 RNAi agent comprises or consists of aduplex having the nucleobase sequences of the sense strand and theantisense strand of any of the sequences in Table 2, Table 3, Table 4,or Table 5.

Examples of antisense strands containing modified nucleotides areprovided in Table 4. Examples of sense strands containing modifiednucleotides are provided in Table 5. Further examples of antisensestrands and sense strands containing modified nucleotides are providedin Table 3.

As used in Tables 3, 4, and 5, the following notations are used toindicate modified nucleotides, targeting groups, and linking groups:

-   -   A=adenosine-3′-phosphate;    -   C=cytidine-3′-phosphate;    -   G=guanosine-3′-phosphate;    -   U=uridine-3′-phosphate    -   I=inosine-3′-phosphate    -   n=any 2′-OMe modified nucleotide    -   a=2′-O-methyladenosine-3′-phosphate    -   as=2′-O-methyladenosine-3′-phosphorothioate    -   c=2′-O-methylcytidine-3′-phosphate    -   cs=2′-O-methylcytidine-3′-phosphorothioate    -   g=2′-O-methylguanosine-3′-phosphate    -   gs=2′-O-methylguanosine-3′-phosphorothioate    -   t=2′-O-methyl-5-methyluridine-3′-phosphate    -   ts=2′-O-methyl-5-methyluridine-3′-phosphorothioate    -   u=2′-O-methyluridine-3′-phosphate    -   us=2′-O-methyluridine-3′-phosphorothioate    -   i=2′-O-methylinosine-3′-phosphate    -   is=2′-O-methylinosine-3′-phosphorothioate    -   Nf=any 2′-fluoro modified nucleotide    -   Af=2′-fluoroadenosine-3′-phosphate    -   Afs=2′-fluoroadenosine-3′-phosporothioate    -   Cf=2′-fluorocytidine-3′-phosphate    -   Cfs=2′-fluorocytidine-3′-phosphorothioate    -   Gf=2′-fluoroguanosine-3′-phosphate    -   Gfs=2′-fluoroguanosine-3′-phosphorothioate    -   If=2′-fluoroinosine-3′-phosphate    -   Ifs=2′-fluoroinosine-3′-phosphorothioate    -   Tf=2′-fluoro-5′-methyluridine-3′-phosphate    -   Tfs=2′-fluoro-5′-methyluridine-3′-phosphorothioate    -   Uf=2′-fluorouridine-3′-phosphate    -   Ufs=2′-fluorouridine-3′-phosphorothioate    -   dN=any 2′-deoxyribonucleotide    -   dA=2′-deoxyadenosine-3′-phosphate    -   dAs=2′-deoxyadenosine-3′-phosphorothioate    -   dC=2′-deoxycytidine-3′-phosphate    -   dCs=2′-deoxycytidine-3′-phosphorothioate    -   dG=2′-deoxyguanosine-3′-phosphate    -   dGs=2′-deoxyguanosine-3′-phosphorothioate    -   dT=2′-deoxythymidine-3′-phosphate    -   dTs=2′-deoxythymidine-3′-phosphorothioate    -   dU=2′-deoxyuridine-3′-phosphate    -   dUs=2′-deoxyuridine-3′-phosphorothioate    -   N_(UNA)=2′,3′-seco nucleotide mimics (unlocked nucleobase        analogs)-3′-Phosphate    -   N_(UNA)s=2′,3′-seco nucleotide mimics (unlocked nucleobase        analogs)-3′-phosphorothioate    -   A_(UNA)=2′,3′-seco-adenosine-3′-phosphate    -   A_(UNAS)=2′,3′-seco-adenosine-3′-phosphorothioate    -   C_(UNA)=2′,3′-seco-cytidine-3′-phosphate    -   C_(UNAS)=2′,3′-seco-cytidine-3′-phosphorothioate    -   G_(UNA)=2′,3′-seco-guanosine-3′-phosphate    -   G_(UNA)s=2′,3′-seco-guanosine-3′-phosphorothioate    -   U_(UNA)=2′,3′-seco-uridine-3′-phosphate    -   U_(UNA)s=2′,3′-seco-uridine-3′-phosphorothioate    -   a_2N=see Table 7    -   a_2Ns=see Table 7    -   pu_2N=see Table 7    -   pu_2Ns=see Table 7    -   N_(LNA)=locked nucleotide    -   Nf_(ANA)=2′-F-Arabino nucleotide    -   NM=2′-O-methoxyethyl nucleotide    -   AM=2′-O-methoxyethyladenosine-3′-phosphate    -   AMs=2′-O-methoxyethyladenosine-3′-phosphorothioate    -   GM=2′-O-methoxyethylguanosine-3′-phosphate    -   GMs=2′-O-methoxyethylguanosine-3′-phosphorothioate    -   TM=2′-O-methoxyethylthymidine-3′-phosphate    -   TMs=2′-O-methoxyethylthymidine-3′-phosphorothioate    -   mCM=see Table 7    -   mCMs=see Table 7    -   R=ribitol    -   (invdN)=any inverted deoxyribonucleotide (3′-3′ linked        nucleotide)    -   (invAb)=inverted (3′-3′ linked) abasic deoxyribonucleotide, see        Table 7    -   (invAb)s=inverted (3′-3′ linked) abasic        deoxyribonucleotide-5′-phosphorothioate, see Table 7    -   (invn)=any inverted 2′-OMe nucleotide (3′-3′ linked nucleotide)    -   s=phosphorothioate linkage    -   sp=see Table 7    -   D2u=see Table 7    -   pD2u=see Table 7    -   vpdN=vinyl phosphonate deoxyribonucleotide    -   (5Me-Nf)=5′-Me, 2′-fluoro nucleotide    -   cPrp=cyclopropyl phosphonate, see Table 7    -   epTcPr=see Table 7    -   epTM=see Table 7

As the person of ordinary skill in the art would readily understand,unless otherwise indicated by the sequence (such as, for example, by aphosphorothioate linkage “s”), when present in an oligonucleotide, thenucleotide monomers are mutually linked by 5′-3′-phosphodiester bonds.As the person of ordinary skill in the art would clearly understand, theinclusion of a phosphorothioate linkage as shown in the modifiednucleotide sequences disclosed herein replaces the phosphodiesterlinkage typically present in oligonucleotides (see, e.g., FIGS. 1Athrough 1I showing all internucleoside linkages). Further, the person ofordinary skill in the art would readily understand that the terminalnucleotide at the 3′ end of a given oligonucleotide sequence wouldtypically have a hydroxyl (—OH) group at the respective 3′ position ofthe given monomer instead of a phosphate moiety ex vivo. Moreover, asthe person of ordinary skill would readily understand and appreciate,while the phosphorothioate chemical structures depicted herein typicallyshow the anion on the sulfur atom, the inventions disclosed hereinencompass all phosphorothioate tautomers and/or diastereomers (e.g.,where the sulfur atom has a double-bond and the anion is on an oxygenatom). Unless expressly indicated otherwise herein, such understandingsof the person of ordinary skill in the art are used when describing theAPOC3 RNAi agents and compositions of APOC3 RNAi agents disclosedherein.

Certain examples of targeting groups and linking groups used with theAPOC3 RNAi agents disclosed herein are provided below in Table 7. Morespecifically, targeting groups and linking groups include the following,for which their chemical structures are provided below in Table 7:(PAZ), (NAG13), (NAG13)s, (NAG18), (NAG18)s, (NAG24), (NAG24)s, (NAG25),(NAG25)s, (NAG26), (NAG26)s, (NAG27), (NAG27)s, (NAG28), (NAG28)s,(NAG29), (NAG29)s, (NAG30), (NAG30)s, (NAG31), (NAG31)s, (NAG32),(NAG32)s, (NAG33), (NAG33)s, (NAG34), (NAG34)s, (NAG35), (NAG35)s,(NAG36), (NAG36)s, (NAG37), (NAG37)s, (NAG38), (NAG38)s, (NAG39),(NAG39)s. Each sense strand and/or antisense strand can have anytargeting groups or linking groups listed herein, as well as othertargeting or linking groups, conjugated to the 5′ and/or 3′ end of thesequence.

TABLE 3APOC3 RNAi Agent Modified Antisense Strand and Modified Sense Strand DuplexesDUPLEX ID Antisense Sequence Sense Sequence NO.: SEQ ID NO. (5′ → 3′)SEQ ID NO. (5′ → 3′)  56_1 181 uAfaCfaAfcAfAfGfgAfgUfaCfcCfguu 246cgGfgUfaCfUfCfcUfuGfuUfgUfuauu  56_2 182 uAfaCfaAfcAfaGfgAfgUfaCfcCfguu247 cggguaCfUfCfcuuguuguuauu  56_3 183 uAfaCfaAfcAfaGfgAfgUfaCfcCfggu248 ccggguaCfUfCfcuuguuguua  56_4 184 uAfaCfaAfcAfaGfgAfgUfaCfcCfggg 249cccggguaCfUfCfcuuguuguua  56_5 185 uAfaCfaAfcaaggAfgUfaCfcCfggg 250cccggguaCfUfCfcuuguuguua  58_1 186 uGfcAfaCfaAfCfAfaGfgAfgUfaCfcuu 251ggUfaCfuCfCfUfuGfuUfgUfuGfcauu  58_2 187 uGfcAfaCfaAfcAfaGfgAfgUfaCfcuu252 gguacuCfCfUfuguuguugcauu  58_3 188 uGfcAfaCfaAfcAfaGfgAfgUfaCfccu253 ggguacuCfCfUfuguuguugca  58_4 189 uGfcAfaCfaAfcAfaGfgAfgUfaCfccg 254cggguacuCfCfUfuguuguugca  58_5 190 uGfcAfaCfaacaaGfgAfgUfaCfccg 255cggguacuCfCfUfuguuguugca 228_1 191 uAfgCfcAfuCfGfGfuCfaCfcCfaGfcuu 256gcUfgGfgUfGfAfcCfgAfuGfgCfuauu 228_2 192 uAfgCfcAfuCfgGfuCfaCfcCfaGfcuu257 gcugggUfGfAfccgauggcuauu 228_3 193 uAfgCfcAfuCfgGfuCfaCfcCfaGfccu258 ggcugggUfGfAfccgauggcua 228_4 194 uAfgCfcAfuCfgGfuCfaCfcCfaGfccc 259gggcugggUfGfAfccgauggcua 228_5 195 uAfgCfcAfucgguCfaCfcCfaGfccc 260gggcugggUfGfAfccgauggcua 235_1 196 uGfaAfcUfgAfAfGfcCfaUfcGfgUfcuu 261gaCfcGfaUfGfGfcUfuCfaGfuUfcauu 235_2 197 uGfaAfcUfgAfaGfcCfaUfcGfgUfcuu262 gaccgaUfGfGfcuucaguucauu 235_3 198 uGfaAfcUfgAfaGfcCfaUfcGfgUfcau263 ugaccgaUfGfGfcuucaguuca 235_4 199 uGfaAfcUfgAfaGfcCfaUfcGfgUfcac 264gugaccgaUfGfGfcuucaguuca 235_5 200 uGfaAfcUfgaagcCfaUfcGfgUfcac 265gugaccgaUfGfGfcuucaguuca 243_1 201 uCfuUfuCfaGfGfGfaAfcUfgAfaGfcuu 266gcUfuCfaGfUfUfcCfcUfgAfaAfgauu 243_2 202 uCfuUfuCfaGfgGfaAfcUfgAfaGfcuu267 gcuucaGfUfUfcccugaaagauu 243_3 203 uCfuUfuCfaGfgGfaAfcUfgAfaGfccu268 ggcuucaGfUfUfcccugaaaga 243_4 204 uCfuUfuCfaGfgGfaAfcUfgAfaGfcca 269uggcuucaGfUfUfcccugaaaga 243_5 205 uCfuUfuCfagggaAfcUfgAfaGfcca 270uggcuucaGfUfUfcccugaaaga 260_1 206 uAfaCfgGfuGfCfUfcCfaGfuAfgUfcuu 271gaCfuAfcUfGfGfaGfcAfcCfgUfuauu 260_2 207 uAfaCfgGfuGfcUfcCfaGfuAfgUfcuu272 gacuacUfGfGfagcaccguuauu 260_3 208 uAfaCfgGfuGfcUfcCfaGfuAfgUfcuu273 agacuacUfGfGfagcaccguua 260_4 209 uAfaCfgGfuGfcUfcCfaGfuAfgUfcuu 274aagacuacUfGfGfagcaccguua 260_5 210 uAfaCfgGfugcucCfaGfuAfgUfcuu 275aagacuacUfGfGfagcaccguua 261_1 211 uUfaAfcGfgUfGfCfuCfcAfgUfaGfuuu 276acUfaCfuGfGfAfgCfaCfcGfuUfaauu 261_2 212 uUfaAfcGfgUfgCfuCfcAfgUfaGfuuu277 acuacuGfGfAfgcaccguuaauu 261_3 213 uUfaAfcGfgUfgCfuCfcAfgUfaGfucu278 gacuacuGfGfAfgcaccguuaa 261_4 214 uUfaAfcGfgUfgCfuCfcAfgUfaGfucu 279agacuacuGfGfAfgcaccguuaa 261_5 215 uUfaAfcGfgugcuCfcAfgUfaGfucu 280agacuacuGfGfAfgcaccguuaa 270_1 216 uAfcUfuGfuCfCfUfuAfaCfgGfuGfcuu 281gcAfcCfgUfUfAfaGfgAfcAfaGfuauu 270_2 217 uAfcUfuGfuCfcUfuAfaCfgGfuGfcuu282 gcaccgUfUfAfaggacaaguauu 270_3 218 uAfcUfuGfuCfcUfuAfaCfgGfuGfcuu283 agcaccgUfUfAfaggacaagua 270_4 219 uAfcUfuGfuCfcUfuAfaCfgGfuGfcuc 284gagcaccgUfUfAfaggacaagua 270_5 220 uAfcUfuGfuccuuAfaCfgGfuGfcuc 285gagcaccgUfUfAfaggacaagua 272_1 221 uGfaAfcUfuGfUfCfcUfuAfaCfgGfuuu 286acCfgUfuAfAfGfgAfcAfaGfuUfcauu 272_2 222 uGfaAfcUfuGfuCfcUfuAfaCfgGfuuu287 accguuAfAfGfgacaaguucauu 272_3 223 uGfaAfcUfuGfuCfcUfuAfaCfgGfugu288 caccguuAfAfGfgacaaguuca 272_4 224 uGfaAfcUfuGfuCfcUfuAfaCfgGfugc 289gcaccguuAfAfGfgacaaguuca 272_5 225 uGfaAfcUfuguccUfuAfaCfgGfugc 290gcaccguuAfAfGfgacaaguuca 273_1 226 uAfgAfaCfuUfGfUfcCfuUfaAfcGfguu 291ccGfuUfaAfGfGfaCfaAfgUfuCfuauu 273_2 227 uAfgAfaCfuUfgUfcCfuUfaAfcGfguu292 ccguuaAfGfGfacaaguucuauu 273_3 228 uAfgAfaCfuUfgUfcCfuUfaAfcGfguu293 accguuaAfGfGfacaaguucua 273_4 229 uAfgAfaCfuUfgUfcCfuUfaAfcGfgug 294caccguuaAfGfGfacaaguucua 273_5 230 uAfgAfaCfuugucCfuUfaAfcGfgug 295caccguuaAfGfGfacaaguucua 349_1 231 uGfgAfcUfuGfGfGfgUfaUfuGfaGfguu 296ccUfcAfaUfAfCfcCfcAfaGfuCfcauu 349_2 232 uGfgAfcUfuGfgGfgUfaUfuGfaGfguu297 ccucaaUfAfCfcccaaguccauu 349_3 233 uGfgAfcUfuGfgGfgUfaUfuGfaGfguu298 accucaaUfAfCfcccaagucca 349_4 234 uGfgAfcUfuGfgGfgUfaUfuGfaGfguc 299gaccucaaUfAfCfcccaagucca 349_5 235 uGfgAfcUfuggggUfaUfuGfaGfguc 300gaccucaaUfAfCfcccaagucca 434_1 236 uGfaGfaAfuAfCfUfgUfcCfcUfuUfuuu 301aaAfaGfgGfAfCfaGfuAfuUfcUfcauu 434_2 237 uGfaGfaAfuAfcUfgUfcCfcUfuUfuuu302 aaaaggGfAfCfaguauucucauu 434_3 238 uGfaGfaAfuAfcUfgUfcCfcUfuUfuau303 uaaaaggGfAfCfaguauucuca 434_4 239 uGfaGfaAfuAfcUfgUfcCfcUfuUfuaa 304uuaaaaggGfAfCfaguauucuca 434_5 240 uGfaGfaAfuacugUfcCfcUfuUfuaa 305uuaaaaggGfAfCfaguauucuca 437_1 241 uAfcUfgAfgAfAfUfaCfuGfuCfcCfuuu 306agGfgAfcAfGfUfaUfuCfuCfaGfuauu 437_2 242 uAfcUfgAfgAfaUfaCfuGfuCfcCfuuu307 agggacAfGfUfauucucaguauu 437_3 243 uAfcUfgAfgAfaUfaCfuGfuCfcCfuuu308 aagggacAfGfUfauucucagua 437_4 244 uAfcUfgAfgAfaUfaCfuGfuCfcCfuuu 309aaagggacAfGfUfauucucagua 437_5 245 uAfcUfgAfgaauaCfuGfuCfcCfuuu 310aaagggacAfGfUfauucucagua

TABLE 4 APOC3 RNAi Agent Antisense Strand SequencesUnderlying Base Sequence Antisense (5′ → 3′) StrandModified Antisense Strand SEQ ID (Shown as an Unmodified ID: (5′ → 3′)NO. Nucleotide Sequence) SEQ ID NO. AM06203-ASusAfscsUfuGfuCfcUfuAfaCfgGfuGfcusu 311 UACUUGUCCUUAACGGUGCUU 603AM06204-AS usAfscsUfuGfuCfcUfuAfaCfgGfuGfcusc 312 UACUUGUCCUUAACGGUGCUC604 AM06205-AS asAfscsUfuGfuCfcUfuAfaCfgGfuGfcusu 313AACUUGUCCUUAACGGUGCUU 655 AM06210-AS usGfsgsAfcUfuGfgGfgUfaUfuGfaGfgusu314 UGGACUUGGGGUAUUGAGGUU 610 AM06211-ASusGfsgsAfcUfuGfgGfgUfaUfuGfaGfgusc 315 UGGACUUGGGGUAUUGAGGUC 611AM06214-AS usCfsusUfuCfaGfgGfaAfcUfgAfaGfcusu 316 UCUUUCAGGGAACUGAAGCUU597 AM06215-AS usCfsusUfuCfaGfgGfaAfcUfgAfaGfccsu 317UCUUUCAGGGAACUGAAGCCU 598 AM06218-AS usGfsasAfcUfgAfaGfcCfaUfcGfgUfcusu318 UGAACUGAAGCCAUCGGUCUU 594 AM06219-ASusGfsasAfcUfgAfaGfcCfaUfcGfgUfcasc 319 UGAACUGAAGCCAUCGGUCAC 596AM06262-AS usGfsasGfaAfuAfcUfgUfcCfcUfuUfugsg 320 UGAGAAUACUGUCCCUUUUGG656 AM06263-AS usGfsasGfaAfuAfcUfgUfcCfcUfuUfugcsg 321UGAGAAUACUGUCCCUUUUGCG 657 AM06266-AS usAfsasCfgGfuGfcUfcCfaGfuAfgUfcusu322 UAACGGUGCUCCAGUAGUCUU 500 AM06267-ASusAfsasCfgGfuGfcUfcCfaGfuAfgUfcgsu 323 UAACGGUGCUCCAGUAGUCGU 658AM06272-AS usAfscsUfgAfgAfaUfaCfuGfuCfcCfuusu 324 UACUGAGAAUACUGUCCCUUU615 AM06273-AS usAfscsUfgAfgAfaUfaCfuGfuCfcCfugsu 325UACUGAGAAUACUGUCCCUGU 659 AM06276-AS usUfsasAfcGfgUfgCfuCfcAfgUfaGfucsu326 UUAACGGUGCUCCAGUAGUCU 602 AM06277-ASusUfsasAfcGfgUfgCfuCfcAfgUfaGfgcsu 327 UUAACGGUGCUCCAGUAGGCU 660AM06309-AS usAfsgsCfcAfuCfgGfuCfaCfcCfaGfcusu 328 UAGCCAUCGGUCACCCAGCUU591 AM06310-AS asAfsgsCfcAfuCfgGfuCfaCfcCfaGfcusu 329AAGCCAUCGGUCACCCAGCUU 661 AM06314-AS usAfsgsAfaCfuUfgUfcCfuUfaAfcGfgusu330 UAGAACUUGUCCUUAACGGUU 608 AM06315-ASusAfsgsAfaCfuUfgUfcCfuUfaAfcGfgusg 331 UAGAACUUGUCCUUAACGGUG 609AM06318-AS usGfsasAfcUfuGfuCfcUfuAfaCfgGfuusu 332 UGAACUUGUCCUUAACGGUUU65 AM06319-AS asGfsasAfcUfuGfuCfcUfuAfaCfgGfuusu 333AGAACUUGUCCUUAACGGUUU 662 AM06320-AS usGfsasAfcUfuGfuCfcUfuAfaCfgGfugsc334 UGAACUUGUCCUUAACGGUGC 607 AM06324-ASusGfscsAfaCfaAfcAfaGfgAfgUfaCfcusu 335 UGCAACAACAAGGAGUACCUU 588AM06325-AS usGfscsAfaCfaAfcAfaGfgAfgUfaCfccsg 336 UGCAACAACAAGGAGUACCCG590 AM06328-AS usAfsasCfaAfcAfaGfgAfgUfaCfcCfgusu 337UAACAACAAGGAGUACCCGUU 585 AM06330-AS usGfscsAfcUfgAfgAfaUfaCfuGfuCfccusu338 UGCACUGAGAAUACUGUCCCUU 663 AM06331-ASasGfscsAfcUfgAfgAfaUfaCfuGfuCfccusu 339 AGCACUGAGAAUACUGUCCCUU 664AM06469-AS cPrpusAfscsUfuGfuCfcUfuAfaCfgGfuGfcusu 340UACUUGUCCUUAACGGUGCUU 603 AM06471-AS asAfscsUfuGfuCfcUfuAfaCfgGfuGfcusc341 AACUUGUCCUUAACGGUGCUC 666 AM06472-AS usAfscsUfuGfuCfcUfuAfaCfgGfugsc342 UACUUGUCCUUAACGGUGC 667 AM06475-ASusAfscsUfuGfuCfcUfuAfaCfgGfuGfcucsc 343 UACUUGUCCUUAACGGUGCUCC 668AM06476-AS usAfscsUfuGfuCfcUfuAfaCfgGfuGfcucusu 344UACUUGUCCUUAACGGUGCUCUU 669 AM06477-ASusAfscsUfuGfuCfcUfuAfaCfgGfuGfcuccsa 345 UACUUGUCCUUAACGGUGCUCCA 670AM06478-AS asAfscsUfuGfuCfcUfuAfaCfgGfugsc 346 AACUUGUCCUUAACGGUGC 671AM06481-AS asAfscsUfuGfuCfcUfuAfaCfgGfuGfcucsc 347AACUUGUCCUUAACGGUGCUCC 672 AM06507-AS usGfsasGfaAfuAfcUfgUfcCfcUfuUfuusu348 UGAGAAUACUGUCCCUUUUUU 612 AM06509-ASusGfsasGfaAfuAfcUfgUfcCfcUfuUfugsu 349 UGAGAAUACUGUCCCUUUUGU 673AM06511-AS usGfsaGfaAfuAfcUfgUfcCfcUfuUfugsg 350 UGAGAAUACUGUCCCUUUUGG656 AM06513-AS asGfsasGfaAfuAfcUfgUfcCfcUfuUfugsg 351AGAGAAUACUGUCCCUUUUGG 674 AM06514-AS usGfscsAfaCfaAfcAfaGfgAfgUfaCfsc352 UGCAACAACAAGGAGUACC 675 AM06517-AS usGfscsAfaCfaacaaGfgAfgUfaCfccsu353 UGCAACAACAAGGAGUACCCU 589 AM06518-ASusGfscsAfaCfaacaaGfgAfgUfaCfcusu 354 UGCAACAACAAGGAGUACCUU 588AM06519-AS usGfscsaacaAfcAfaGfgAfguaccusu 355 UGCAACAACAAGGAGUACCUU 588AM06521-AS usGfcAfaCfaAfcAfaGfgAfgUfaCfcusu 356 UGCAACAACAAGGAGUACCUU588 AM06523-AS asGfscsAfaCfaAfcAfaGfgAfgUfaCfcusu 357AGCAACAACAAGGAGUACCUU 676 AM06712-AS usCfsusGfaAfgccauCfgGfuCfaCfcCfsa358 UCUGAAGCCAUCGGUCACCCA 677 AM06714-ASasCfsusGfaAfgccauCfgGfuCfaCfcCfsa 359 ACUGAAGCCAUCGGUCACCCA 678AM06716-AS usGfsgsAfaCfugaagCfcAfuCfgGfuCfsa 360 UGGAACUGAAGCCAUCGGUCA679 AM06718-AS usGfsgsAfaCfugaagCfcAfuCfgGfuCfsc 361UGGAACUGAAGCCAUCGGUCC 680 AM06720-AS usUfscsUfuUfcagggAfaCfuGfaAfgCfsc362 UUCUUUCAGGGAACUGAAGCC 681 AM06722-ASusUfsusAfaCfggugcUfcCfaGfuAfgUfsc 363 UUUAACGGUGCUCCAGUAGUC 682AM06724-AS usCfscsUfuAfacgguGfcUfcCfaGfuAfsg 364 UCCUUAACGGUGCUCCAGUAG683 AM06726-AS usUfscsCfuUfaacggUfgCfuCfcAfgUfsa 365UUCCUUAACGGUGCUCCAGUA 684 AM06728-AS usUfscsCfuUfaacggUfgCfuCfcAfgUfsc366 UUCCUUAACGGUGCUCCAGUC 685 AM06730-ASusAfscsUfuGfuCfcUfuAfaCfgGfuGfcUfsc 367 UACUUGUCCUUAACGGUGCUC 604AM06732-AS asAfscsUfuGfuCfcUfuAfaCfgGfuGfcsUfsc 368AACUUGUCCUUAACGGUGCUC 666 AM06734-AS usUfsgsAfgGfucucaGfgCfaGfcCfaCfsu369 UUGAGGUCUCAGGCAGCCACU 686 AM06736-ASusUfsasUfuGfaGfgUfcUfcAfgGfcAfgCfsc 370 UUAUUGAGGUCUCAGGCAGCC 687AM06738-AS usGfsusAfuUfgAfgGfuCfuCfaGfgCfaGfsc 371 UGUAUUGAGGUCUCAGGCAGC688 AM06740-AS usCfsasCfuGfaGfaAfuAfcUfgUfcCfcUfsu 372UCACUGAGAAUACUGUCCCUU 689 AM06741-AS usCfsasCfuGfagaauAfcUfgUfcCfcUfsu373 UCACUGAGAAUACUGUCCCUU 689 AM06743-ASusCfsasCfuGfagaauAfcUfgUfcCfcGfsu 4 UCACUGAGAAUACUGUCCCGU 5 AM06745-ASusCfsusUfuUfaAfgCfaAfcCfuAfcAfgGfsg 374 UCUUUUAAGCAACCUACAGGG 690AM06780-AS usGfsasGfaAfuAfcUfgUfcCfcUfuUfucsc 375 UGAGAAUACUGUCCCUUUUCC691 AM06783-AS usCfsasCfuGfagaauAfcUfgUfcCfcUfsc 2 UCACUGAGAAUACUGUCCCUC3 AM06784-AS usUfsasUfuGfaggucUfcAfgGfcAfgCfsc 376 UUAUUGAGGUCUCAGGCAGCC687 AM06786-AS usGfsasGfaAfuAfcUfgUfcCfcUfuUfgcsc 13UGAGAAUACUGUCCCUUUGCC 14 AM06862-AS usGfsasGfaAfuAfcUfgUfcCfcUfuUfuCfsc377 UGAGAAUACUGUCCCUUUUCC 691 AM06865-ASusGfsasGfaAfuAfcUfgUfcCfcUfuUfucsu 378 UGAGAAUACUGUCCCUUUUCU 692AM06868-AS usUfscsUfuGfuCfcAfgCfuUfuAfuUfgGfsg 379 UUCUUGUCCAGCUUUAUUGGG693 AM06870-AS usUfscsUfuGfuCfcAfgCfuUfuAfuUfgGfsc 7UUCUUGUCCAGCUUUAUUGGC 8 AM06872-AS usAfsgsUfcUfuUfcAfgGfgAfaCfuGfaAfsg380 UAGUCUUUCAGGGAACUGAAG 694 AM06874-ASusAfsgsUfcUfuUfcAfgGfgAfaCfuGfaAfsc 381 UAGUCUUUCAGGGAACUGAAC 695AM06876-AS asGfsasAfuAfcUfgUfcCfcUfuUfuAfaGfsc 11 AGAAUACUGUCCCUUUUAAGC12 AM06908-AS usCfsasCfuGfagaauAfcUfgUfcCfcusu 382 UCACUGAGAAUACUGUCCCUU689 AM06928-AS usCfsasCfuGfagaauAfcUfgUfcCfgusu 383UCACUGAGAAUACUGUCCGUU 696 AM06951-AS usAfsgsUfcUfuUfcAfgGfgAfaCfuGfaCfsg384 UAGUCUUUCAGGGAACUGACG 697 AM06953-ASusAfsgsUfcUfuUfcAfgGfgAfaCfuGfaGfsg 385 UAGUCUUUCAGGGAACUGAGG 698AM06956-AS usAfsgsUfcUfuUfcAfgGfgAfaCfuGfaCfsc 386 UAGUCUUUCAGGGAACUGACC699 AM06958-AS usAfsgsUfcUfuUfcAfgGfgAfaCfuGfaGfsc 387UAGUCUUUCAGGGAACUGAGC 700 AM06961-AS asGfsasAfuAfcUfgUfcCfcUfuUfuAfgGfsc388 AGAAUACUGUCCCUUUUAGGC 701 AM06963-ASasGfsasAfuAfcUfgUfcCfcUfuUfuAfaGfsg 389 AGAAUACUGUCCCUUUUAAGG 702AM06988-AS asGfsasAfuAfcUfgUfcCfcUfuUfuAfgGfsg 9 AGAAUACUGUCCCUUUUAGGG10 AM07179-AS usGfscsAfaCfA_(UNA)acaaGfgAfgUfaCfccsu 390UGCAACAACAAGGAGUACCCU 589 AM07182-ASusAfsgsUfcUfU_(UNA)UfcAfgGfgAfaCfuGfaAfsg 391 UAGUCUUUCAGGGAACUGAAG 694AM07185-AS asGfsasAfuAfC_(UNA)UfgUfcCfcUfuUfuAfaGfsc 392AGAAUACUGUCCCUUUUAAGC 12 AM07188-ASusGfsasGfaAfU_(UNA)AfcUfgUfcCfcUfuUfgcsc 393 UGAGAAUACUGUCCCUUUGCC 14AM07190-AS usCfsasCfuGfA_(UNA)gaauAfcUfgUfcCfcUfsc 394UCACUGAGAAUACUGUCCCUC 3 AM07193-ASusUfscsUfuGfU_(UNA)CfcAfgCfuUfuAfuUfgGfsc 395 UUCUUGUCCAGCUUUAUUGGC 8AM07518-AS asGfsasAfuAfcUfgUfcCfcUfuUfuAfgGfsu 396 AGAAUACUGUCCCUUUUAGGU707 AM07520-AS asGfsasAfuAfcUfgUfcCfcUfuUfuAfcGfsc 397AGAAUACUGUCCCUUUUACGC 708 AM07522-AS asGfsasAfuAfcUfgUfcCfcUfuUfuAfgAfsc398 AGAAUACUGUCCCUUUUAGAC 709 AM07524-AS usCfsascugagaauAfcUfgUfcCfcUfsc6 UCACUGAGAAUACUGUCCCUC 3 AM07600-AS asGfsasauacugucCfcUfuUfuAfgGfsc 399AGAAUACUGUCCCUUUUAGGC 701 AM07645-AS usUfscsuuguccagCfuUfuAfuUfgGfsc 400UUCUUGUCCAGCUUUAUUGGC 8 AM07750-AS usCfsasCfuGfagaauAfcUfgUfcCfcUfsg 401UCACUGAGAAUACUGUCCCUG 710 AM07753-AS usCfsasCfuGfagaauAfcUfgUfcCfcCfsu402 UCACUGAGAAUACUGUCCCCU 711 AM07755-ASusCfsA_(UNA)sCfuGfagaauAfcUfgUfcCfcUfsc 403 UCACUGAGAAUACUGUCCCUC 3AM07756-AS usCfsasC_(UNA)uGfagaauAfcUfgUfcCfcUfsc 404UCACUGAGAAUACUGUCCCUC 3 AM07757-ASusCfsasCfU_(UNA)GfagaauAfcUfgUfcCfcUfsc 405 UCACUGAGAAUACUGUCCCUC 3AM07758-AS usCfsasCfuG_(UNA)agaauAfcUfgUfcCfcUfsc 406UCACUGAGAAUACUGUCCCUC 3 AM07760-AS asGfsusGfcAfuccuuGfgCfgGfuCfuusu 407AGUGCAUCCUUGGCGGUCUUU 712 AM07762-ASasGfsusGfcAfU_(UNA)ccuuGfgCfgGfuCfuusu 408 AGUGCAUCCUUGGCGGUCUUU 712AM07764-AS asGfsusAfgUfcuuucAfgGfgAfaCfuGfsa 409 AGUAGUCUUUCAGGGAACUGA713 AM07765-AS asGfsusAfgUfC_(UNA)uuucAfgGfgAfaCfuGfsa 410AGUAGUCUUUCAGGGAACUGA 713 AM07767-AS usGfsusAfgUfcuuucAfgGfgAfaCfuGfsa411 UGUAGUCUUUCAGGGAACUGA 714 AM07769-ASusCfsusUfaAfcggugCfuCfcAfgUfaGfsu 412 UCUUAACGGUGCUCCAGUAGU 715AM07771-AS usCfscsUfuUfuaagcAfaCfcUfaCfaGfsg 413 UCCUUUUAAGCAACCUACAGG716 AM07773-AS usCfscsUfuUfuaagcAfaCfcUfaCfaGfsc 414UCCUUUUAAGCAACCUACAGC 717 AM07775-AS usAfsgsUfcUfuucagGfgAfaCfuGfaCfsc415 UAGUCUUUCAGGGAACUGACC 699

TABLE 5 APOC3 RNAi Agent Sense Strand Sequences Underlying Base SequenceSEQ (5′ → 3′) SEQ Sense Strand ID (Shown as an Unmodified ID ID:Modified Sense Strand (5′ → 3′) NO. Nucleotide Sequence) NO. AM06206-SS(NAG37)s(invAb)sgcaccgUfUfAfaggacaaguauus(invAb) 416GCACCGUUAAGGACAAGUAUU 718 AM06207-SS(NAG37)s(invAb)sgagcaccgUfUfAfaggacaagus(invdA) 417GAGCACCGUUAAGGACAAGUA 719 AM06208-SS(NAG37)s(invAb)sgcaccgUfUfAfaggacaagus(invdA) 418 GCACCGUUAAGGACAAGUA720 AM06209-SS (NAG37)s(invAb)sgcaccgUfUfAfaggacaaguus(invAb) 419GCACCGUUAAGGACAAGUU 721 AM06212-SS(NAG37)s(invAb)sccucaaUfAfCfcccaaguccs(invdA) 420 CCUCAAUACCCCAAGUCCA722 AM06213-SS (NAG37)s(invAb)sgaccucaaUfAfCfcccaaguccs(invdA) 421GACCUCAAUACCCCAAGUCCA 723 AM06216-SS(NAG37)s(invAb)sgcuucaGfUfUfcccugaaags(invdA) 422 GCUUCAGUUCCCUGAAAGA724 AM06217-SS (NAG37)s(invAb)sggcuucaGfUfUfcccugaaags(invdA) 423GGCUUCAGUUCCCUGAAAGA 725 AM06220-SS(NAG37)s(invAb)sgaccgaUfGfGfcuucaguucs(invdA) 424 GACCGAUGGCUUCAGUUCA726 AM06221-SS (NAG37)s(invAb)sgugaccgaUfGfGfcuucaguucs(invdA) 425GUGACCGAUGGCUUCAGUUCA 727 AM06264-SS(NAG37)s(invAb)sccaaaaggGfAfCfaguauucucs(invdA) 426CCAAAAGGGACAGUAUUCUCA 728 AM06265-SS(NAG37)scsgcaaaaggGfAfCfaguauucucs(invdA) 427 CGCAAAAGGGACAGUAUUCUCA 729AM06268-SS (NAG37)s(invAb)sgacuacUfGfGfagcaccguus(invdA) 428GACUACUGGAGCACCGUUA 730 AM06269-SS(NAG37)s(invAb)sgacuacUfGfGfagcacuguus(invdA) 429 GACUACUGGAGCACUGUUA731 AM06270-SS (NAG37)s(invAb)scgacuacUfGfGfagcaccguus(invdA) 430CGACUACUGGAGCACCGUUA 732 AM06271-SS(NAG37)s(invAb)sgacuacUfGfGfagcaucguus(invdA) 431 GACUACUGGAGCAUCGUUA733 AM06274-SS (NAG37)s(invAb)sagggacAfGfUfauucucagus(invdA) 432AGGGACAGUAUUCUCAGUA 734 AM06275-SS(NAG37)s(invAb)scagggacAfGfUfauucucagus(invdA) 433 CAGGGACAGUAUUCUCAGUA735 AM06278-SS (NAG37)s(invAb)sgacuacuGfGfAfgcaccguuas(invdA) 434GACUACUGGAGCACCGUUAA 736 AM06279-SS(NAG37)s(invAb)sgccuacuGfGfAfgcaccguuas(invdA) 435 GCCUACUGGAGCACCGUUAA737 AM06280-SS (NAG37)s(invAb)sgccuacuGfGfAfgcacuguuas(invdA) 436GCCUACUGGAGCACUGUUAA 738 AM06311-SS(NAG37)s(invAb)sgcugggUfGfAfccgauggcus(invdA) 437 GCUGGGUGACCGAUGGCUA739 AM06312-SS (NAG37)s(invAb)sgcugggUfGfAfccgauggcuus(invAb) 438GCUGGGUGACCGAUGGCUU 740 AM06313-SS(NAG37)s(invAb)sgcugggUfGfAfccgaugacus(invdA) 439 GCUGGGUGACCGAUGACUA741 AM06316-SS (NAG37)s(invAb)sccguuaAfGfGfacaaguucus(invdA) 440CCGUUAAGGACAAGUUCUA 742 AM06317-SS(NAG37)s(invAb)scaccguuaAfGfGfacaaguucus(invdA) 441CACCGUUAAGGACAAGUUCUA 743 AM06321-SS(NAG37)s(invAb)saccguuAfAfGfgacaaguucs(invdA) 442 ACCGUUAAGGACAAGUUCA744 AM06322-SS (NAG37)s(invAb)saccguuAfAfGfgacaaguucus(invAb) 443ACCGUUAAGGACAAGUUCU 745 AM06323-SS(NAG37)s(invAb)sgcaccguuAfAfGfgacaaguucs(invdA) 444GCACCGUUAAGGACAAGUUCA 746 AM06326-SS(NAG37)s(invAb)sgguacuCfCfUfuguuguugcs(invdA) 445 GGUACUCCUUGUUGUUGCA747 AM06327-SS (NAG37)s(invAb)scggguacuCfCfUfuguuguugcs(invdA) 446CGGGUACUCCUUGUUGUUGCA 748 AM06329-SS(NAG37)s(invAb)scggguaCfUfCfcuuguuguus(invdA) 447 CGGGUACUCCUUGUUGUUA749 AM06332-SS (NAG37)s(invAb)sgggacagUfAfUfucucagugcs(invdA) 448GGGACAGUAUUCUCAGUGCA 750 AM06333-SS(NAG37)s(invAb)sgggacagUfAfUfucucagugcus(invAb) 449 GGGACAGUAUUCUCAGUGCU751 AM06470-SS (NAG37)sgscaccgUfUfAfaggacaaguuuus(invAb) 450GCACCGUUAAGGACAAGUUUU 752 AM06473-SS(NAG37)sgsagcaccgUfUfAfaggacaagus(invdA) 451 GAGCACCGUUAAGGACAAGUA 719AM06474-SS (NAG37)sgsgagcaccgUfUfAfaggacaagus(invdA) 452GGAGCACCGUUAAGGACAAGUA 753 AM06479-SS(NAG37)sgsagcaccgUfUfAfaggacaaguus(invAb) 453 GAGCACCGUUAAGGACAAGUU 754AM06480-SS (NAG37)sgsgagcaccgUfUfAfaggacaaguus(invAb) 454GGAGCACCGUUAAGGACAAGUU 755 AM06506-SS(NAG37)s(invAb)saaaaggGfAfCfaguauucucauus(invAb) 455AAAAGGGACAGUAUUCUCAUU 756 AM06508-SS(NAG37)s(invAb)scaaaaggGfAfCfaguauucucs(invdA) 456 CAAAAGGGACAGUAUUCUCA757 AM06510-SS (NAG37)(invAb)ccaaaaggGfAfCfaguauucuc(invdA) 457CCAAAAGGGACAGUAUUCUCA 728 AM06512-SS(NAG37)s(invAb)sccaaaaggGfAfCfaguauucucus(invAb) 458CCAAAAGGGACAGUAUUCUCU 758 AM06515-SS(NAG37)s(invAb)sgguacuCfCfUfuguuguugcauus(invAb) 459GGUACUCCUUGUUGUUGCAUU 759 AM06516-SS(NAG37)s(invAb)sggguacuCfCfUfuguuguugcs(invdA) 460 GGGUACUCCUUGUUGUUGCA760 AM06520-SS (NAG37)(invAb)gguacuCfCfUfuguuguugc(invdA) 461GGUACUCCUUGUUGUUGCA 747 AM06522-SS(NAG37)s(invAb)sgguacuCfCfUfuguuguugcus(invAb) 462 GGUACUCCUUGUUGUUGCU761 AM06711-SS (NAG37)s(invAb)sugggugacCfGfAfuggcuucagas(invAb) 463UGGGUGACCGAUGGCUUCAGA 762 AM06713-SS(NAG37)s(invAb)sugggugacCfGfAfuggcuucagus(invAb) 464UGGGUGACCGAUGGCUUCAGU 763 AM06715-SS(NAG37)s(invAb)sugaccgauGfGfCfuucaguuccas(invAb) 465UGACCGAUGGCUUCAGUUCCA 764 AM06717-SS(NAG37)s(invAb)sggaccgauGfGfCfuucaguuccas(invAb) 466GGACCGAUGGCUUCAGUUCCA 765 AM06719-SS(NAG37)s(invAb)sggcuucagUfUfCfccugaaagaas(invAb) 467GGCUUCAGUUCCCUGAAAGAA 766 AM06721-SS(NAG37)s(invAb)sgacuacugGfAfGfcaccguuaaas(invAb) 468GACUACUGGAGCACCGUUAAA 767 AM06723-SS(NAG37)s(invAb)scuacuggaGfCfAfccguuaaggas(invAb) 469CUACUGGAGCACCGUUAAGGA 768 AM06725-SS(NAG37)s(invAb)suacuggagCfAfCfcguuaaggaas(invAb) 470UACUGGAGCACCGUUAAGGAA 769 AM06727-SS(NAG37)s(invAb)sgacuggagCfAfCfcguuaaggaas(invAb) 471GACUGGAGCACCGUUAAGGAA 770 AM06729-SS(NAG37)s(invAb)sgagcaccgUfUfAfaggacaaguas(invAb) 472GAGCACCGUUAAGGACAAGUA 719 AM06731-SS(NAG37)s(invAb)sgagcaccgUfUfAfaggacaaguus(invAb) 473GAGCACCGUUAAGGACAAGUU 754 AM06733-SS(NAG37)s(invAb)saguggcugCfCfUfgagaccucaas(invAb) 474AGUGGCUGCCUGAGACCUCAA 771 AM06735-SS(NAG37)s(invAb)sggcugccuGfAfGfaccucaauaas(invAb) 475GGCUGCCUGAGACCUCAAUAA 772 AM06737-SS(NAG37)s(invAb)sgcugccugAfGfAfccucaauacas(invAb) 476GCUGCCUGAGACCUCAAUACA 773 AM06739-SS(NAG37)s(invAb)saagggacaGfUfAfuucucagugas(invAb) 477AAGGGACAGUAUUCUCAGUGA 774 AM06742-SS(NAG37)s(invAb)sacgggacaGfUfAfuucucagugas(invAb) 478ACGGGACAGUAUUCUCAGUGA 775 AM06744-SS(NAG37)s(invAb)scccuguagGfUfUfgcuuaaaagas(invAb) 479CCCUGUAGGUUGCUUAAAAGA 776 AM06779-SS(NAG37)s(invAb)sggaaaaggGfAfCfaguauucucas(invAb) 480GGAAAAGGGACAGUAUUCUCA 777 AM06781-SS(NAG37)gsgaaaaggGfAfCfaguauucucas(invAb) 481 GGAAAAGGGACAGUAUUCUCA 777AM06782-SS (NAG37)s(invAb)sgagggacaGfUfAfuucucagugas(invAb) 482GAGGGACAGUAUUCUCAGUGA 21 AM06785-SS(NAG37)s(invAb)sggcaaaggGfAfCfaguauucucas(invAb) 483GGCAAAGGGACAGUAUUCUCA 31 AM06787-SS(NAG37)gsgcaaaggGfAfCfaguauucucas(invAb) 484 GGCAAAGGGACAGUAUUCUCA 31AM06788-SS (NAG37)s(invAb)susgaccgauGfGfCfuucaiuuccas(invAb) 485UGACCGAUGGCUUCAIUUCCA 780 AM06789-SS(NAG37)usgaccgauGfGfCfuucaguuccas(invAb) 486 UGACCGAUGGCUUCAGUUCCA 764AM06790-SS (NAG37)usgaccgauGfGfCfuucaiuuccas(invAb) 487UGACCGAUGGCUUCAIUUCCA 780 AM06791-SS(NAG37)gsagggacaGfUfAfuucucagugas(invAb) 488 GAGGGACAGUAUUCUCAGUGA 21AM06792-SS (NAG37)gsgcugccuGfAfGfaccucaauaas(invAb) 489GGCUGCCUGAGACCUCAAUAA 772 AM06863-SS(NAG37)s(invAb)sgga_2NaaaggGfAfCfaguauucucas(invAb) 490GG(A^(2N))AAAGGGACAGUAUUCUCA 781 AM06864-SS(NAG37)s(invAb)sa_2NgaaaaggGfAfCfaguauucucas(invAb) 491(A^(2N))GAAAAGGGACAGUAUUCUCA 782 AM06866-SS(NAG37)s(invAb)sa_2Na_2NaaaaggGfAfCfaguauucucas(invAb) 492(A^(2N))(A^(2N))AAAAGGGACAGUAUUCUCA 783 AM06867-SS(NAG37)s(invAb)scccaauaaAfGfCfuggacaagaas(invAb) 493CCCAAUAAAGCUGGACAAGAA 784 AM06869-SS(NAG37)s(invAb)sgccaauaaAfGfCfuggacaagaas(invAb) 494GCCAAUAAAGCUGGACAAGAA 23 AM06871-SS(NAG37)s(invAb)scuucaguuCfCfCfugaaagacuas(invAb) 495CUUCAGUUCCCUGAAAGACUA 786 AM06873-SS(NAG37)s(invAb)sguucaguuCfCfCfugaaagacuas(invAb) 496GUUCAGUUCCCUGAAAGACUA 787 AM06875-SS(NAG37)s(invAb)sgcuuaaaaGfGfGfacaguauucus(invAb) 497GCUUAAAAGGGACAGUAUUCU 29 AM06907-SS(NAG37)s(invAb)sgggacaGfUfAfuucucagugauus(invAb) 498GGGACAGUAUUCUCAGUGAUU 789 AM06922-SS(NAG37)s(invAb)sa_2NagggacaGfUfAfuucucagugas(invAb) 499(A^(2N))AGGGACAGUAUUCUCAGUGA 790 AM06923-SS(NAG37)s(invAb)sgagggacaGfUfAfuucucaiugas(invAb) 500GAGGGACAGUAUUCUCAIUGA 791 AM06924-SS(NAG37)s(invAb)sgagggacaGfUfAfuucucaguias(invAb) 501GAGGGACAGUAUUCUCAGUIA 16 AM06925-SS(NAG37)ascgggacaGfUfAfuucucagugas(invAb) 502 ACGGGACAGUAUUCUCAGUGA 775AM06926-SS (NAG37)gsggacaGfUfAfuucucagugauus(invAb) 503GGGACAGUAUUCUCAGUGAUU 789 AM06927-SS(NAG37)s(invAb)scggacaGfUfAfuucucagugauus(invAb) 504CGGACAGUAUUCUCAGUGAUU 793 AM06929-SS(NAG37)gsgcaaaggGfAfCfaGuauucucas(invAb) 505 GGCAAAGGGACAGUAUUCUCA 31AM06932-SS (NAG37)s(invAb)sggcaaagiGfAfCfaguauucucas(invAb) 506GGCAAAGIGACAGUAUUCUCA 794 AM06933-SS(NAG37)s(invAb)sggcaaaigGfAfCfaguauucucas(invAb) 507GGCAAAIGGACAGUAUUCUCA 778 AM06934-SS(NAG37)s(invAb)saggguacuCfCfUfuguuguugcas(invAb) 508AGGGUACUCCUUGUUGUUGCA 795 AM06948-SS(NAG37)s(invAb)scuucaguuCfUfCfugaaagacuas(invAb) 509CUUCAGUUCUCUGAAAGACUA 796 AM06949-SS(NAG37)s(invAb)scuucaguuUfCfCfugaaagacuas(invAb) 510CUUCAGUUUCCUGAAAGACUA 797 AM06950-SS(NAG37)s(invAb)scgucaguuCfCfCfugaaagacuas(invAb) 511CGUCAGUUCCCUGAAAGACUA 798 AM06952-SS(NAG37)s(invAb)sccucaguuCfCfCfugaaagacuas(invAb) 512CCUCAGUUCCCUGAAAGACUA 799 AM06954-SS(NAG37)s(invAb)scgucaguuCfUfCfugaaagacuas(invAb) 513CGUCAGUUCUCUGAAAGACUA 800 AM06955-SS(NAG37)s(invAb)sggucaguuCfCfCfugaaagacuas(invAb) 514GGUCAGUUCCCUGAAAGACUA 801 AM06957-SS(NAG37)s(invAb)sgcucaguuCfCfCfugaaagacuas(invAb) 515GCUCAGUUCCCUGAAAGACUA 802 AM06960-SS(NAG37)s(invAb)sgccuaaaaGfGfGfacaguauucus(invAb) 516GCCUAAAAGGGACAGUAUUCU 803 AM06962-SS(NAG37)s(invAb)sccuuaaaaGfGfGfacaguauucus(invAb) 517CCUUAAAAGGGACAGUAUUCU 804 AM06964-SS(NAG37)s(invAb)sgcuuaaaaGfGfiacaguauucus(invAb) 518GCUUAAAAGGIACAGUAUUCU 805 AM06965-SS(NAG37)s(invAb)sgcuuaaaaGfiGfacaguauucus(invAb) 519GCUUAAAAGIGACAGUAUUCU 779 AM06966-SS(NAG37)s(invAb)sgcuuaaaaiGfGfacaguauucus(invAb) 520GCUUAAAAIGGACAGUAUUCU 785 AM06987-SS(NAG37)s(invAb)scccuaaaaGfGfGfacaguauucus(invAb) 521CCCUAAAAGGGACAGUAUUCU 27 AM07178-SS(NAG37)s(invAb)saggguacuCfCfUfuguuguuicas(invAb) 522AGGGUACUCCUUGUUGUUICA 807 AM07180-SS(NAG37)s(invAb)saggguacuCfCfUfuGuuguugcas(invAb) 523AGGGUACUCCUUGUUGUUGCA 795 AM07181-SS(NAG37)s(invAb)scuucaguuCfCfCfugaaagaiuas(invAb) 524CUUCAGUUCCCUGAAAGAIUA 808 AM07183-SS(NAG37)s(invAb)scuucaguuCfCfCfuGaaagacuas(invAb) 525CUUCAGUUCCCUGAAAGACUA 786 AM07184-SS(NAG37)s(invAb)sgcuuaaaaGfGfGfacaguauuius(invAb) 526GCUUAAAAGGGACAGUAUUIU 809 AM07186-SS(NAG37)s(invAb)sgcuuaaaaGfGfGfaCaguauucus(invAb) 527GCUUAAAAGGGACAGUAUUCU 29 AM07187-SS(NAG37)s(invAb)sggcaaaggGfAfCfaguauucuias(invAb) 528GGCAAAGGGACAGUAUUCUIA 810 AM07189-SS(NAG37)s(invAb)sggcaaaggGfAfCfaGuauucucas(invAb) 529GGCAAAGGGACAGUAUUCUCA 31 AM07191-SS(NAG37)s(invAb)sgagggacaGfUfAfuUcucaguias(invAb) 530GAGGGACAGUAUUCUCAGUIA 16 AM07192-SS(NAG37)s(invAb)sgccaauaaAfGfCfuggacaaiaas(invAb) 531GCCAAUAAAGCUGGACAAIAA 811 AM07194-SS(NAG37)s(invAb)sgccaauaaAfGfCfuGgacaagaas(invAb) 532GCCAAUAAAGCUGGACAAGAA 23 AM07309-SS(NAG37)s(invAb)saggguacuCflfUfuguuguugcas(invAb) 533AGGGUACUCIUUGUUGUUGCA 812 AM07310-SS(NAG37)s(invAb)saggguacuIfCfUfuguuguugcas(invAb) 534AGGGUACUICUUGUUGUUGCA 788 AM07311-SS(NAG37)s(invAb)scuucaguuCfCflfugaaagacuas(invAb) 535CUUCAGUUCCIUGAAAGACUA 813 AM07312-SS(NAG37)s(invAb)scuucaguuCflfCfugaaagacuas(invAb) 536CUUCAGUUCICUGAAAGACUA 792 AM07313-SS(NAG37)s(invAb)scuucaguuIfCfCfugaaagacuas(invAb) 537CUUCAGUUICCUGAAAGACUA 806 AM07314-SS(NAG37)s(invAb)sgcuuaaaaGfGflfacaguauucus(invAb) 538GCUUAAAAGGIACAGUAUUCU 805 AM07315-SS(NAG37)s(invAb)sgcuuaaaaGflfGfacaguauucus(invAb) 539GCUUAAAAGIGACAGUAUUCU 817 AM07316-SS(NAG37)s(invAb)sgcuuaaaaIfGfGfacaguauucus(invAb) 540GCUUAAAAIGGACAGUAUUCU 824 AM07317-SS(NAG37)s(invAb)sggcaaaggGfAflfaguauucucas(invAb) 541GGCAAAGGGAIAGUAUUCUCA 814 AM07318-SS(NAG37)s(invAb)sggcaaaggIfAfCfaguauucucas(invAb) 542GGCAAAGGIACAGUAUUCUCA 794 AM07319-SS(NAG37)s(invAb)sgagggacaIfUfAfuucucaguias(invAb) 543GAGGGACAIUAUUCUCAGUIA 815 AM07320-SS(NAG37)s(invAb)sgccaauaaAfGflfuggacaagaas(invAb) 544GCCAAUAAAGIUGGACAAGAA 816 AM07321-SS(NAG37)s(invAb)sgccaauaaAfIfCfuggacaagaas(invAb) 545GCCAAUAAAICUGGACAAGAA 25 AM07515-SS(NAG37)s(invAb)sgccuaaaaGfGfiacaguauucus(invAb) 546GCCUAAAAGGIACAGUAUUCU 818 AM07516-SS(NAG37)s(invAb)sgccuaaaaGfGflfacaguauucus(invAb) 547GCCUAAAAGGIACAGUAUUCU 818 AM07517-SS(NAG37)s(invAb)saccuaaaaGfGfGfacaguauucus(invAb) 548ACCUAAAAGGGACAGUAUUCU 819 AM07519-SS(NAG37)s(invAb)sgcguaaaaGfGfGfacaguauucus(invAb) 549GCGUAAAAGGGACAGUAUUCU 820 AM07521-SS(NAG37)s(invAb)sgucuaaaaGfGfGfacaguauucus(invAb) 550GUCUAAAAGGGACAGUAUUCU 821 AM07523-SS(NAG37)s(invAb)sgagggacaGfUfAfuUcucagugas(invAb) 551GAGGGACAGUAUUCUCAGUGA 21 AM07525-SS(NAG37)s(invAb)sgggacaGfUfAfuucucaguiauus(invAb) 552GGGACAGUAUUCUCAGUIAUU 822 AM07526-SS(NAG37)s(invAb)sgccaauaaAfGfCfudGgacaagaas(invAb) 553GCCAAUAAAGCUGGACAAGAA 23 AM07598-SS(NAG37)s(invAb)sgccuaaaaGfgGfaCfaguauucus(invAb) 554GCCUAAAAGGGACAGUAUUCU 803 AM07599-SS(NAG37)s(invAb)sgccuaaaaGfgIfaCfaguauucus(invAb) 555GCCUAAAAGGIACAGUAUUCU 818 AM07601-SS(NAG37)s(invAb)sgagggacaGfuAfuUfcucagugas(invAb) 556GAGGGACAGUAUUCUCAGUGA 21 AM07602-SS(NAG37)s(invAb)sgagggacaGfuAfuUfcucaguias(invAb) 557GAGGGACAGUAUUCUCAGUIA 16 AM07644-SS(NAG37)s(invAb)sgcCfaAfuAfaAfGfCfuggacaagaas(invAb) 558GCCAAUAAAGCUGGACAAGAA 23 AM07646-SS(NAG37)s(invAb)sgcCfaAfuAfaAfgCfuggacaagaas(invAb) 559GCCAAUAAAGCUGGACAAGAA 23 AM07647-SS(NAG37)s(invAb)sgcCfaAfUfAfaAfGfCfuggacaagaas(invAb) 560GCCAAUAAAGCUGGACAAGAA 23 AM07648-SS(NAG37)s(invAb)sgccaauaAMAfGfCfuggacaagaas(invAb) 561GCCAAUAAAGCUGGACAAGAA 23 AM07649-SS(NAG37)s(invAb)sgccAMauaaAfGfCfuggacaagaas(invAb) 562GCCAAUAAAGCUGGACAAGAA 23 AM07650-SS(NAG37)s(invAb)sgcCfaAfuAfaAfIfCfuggacaagaas(invAb) 563GCCAAUAAAICUGGACAAGAA 25 AM07651-SS(NAG37)s(invAb)sgcCfaAfUfAfaAfIfCfuggacaagaas(invAb) 564GCCAAUAAAICUGGACAAGAA 25 AM07652-SS(NAG37)s(invAb)sgccaauaaAfiCfuggacaagaas(invAb) 565GCCAAUAAAICUGGACAAGAA 25 AM07653-SS(NAG37)s(invAb)sgcCfaAfuAfaAfGfCfuigacaagaas(invAb) 566GCCAAUAAAGCUIGACAAGAA 823 AM07654-SS(NAG37)s(invAb)sgcCfaAfuAfaAfGfCfugiacaagaas(invAb) 567GCCAAUAAAGCUGIACAAGAA 836 AM07655-SS(NAG37)s(invAb)sgcCfuAfaAfaGfGfGfacaguauucus(invAb) 568GCCUAAAAGGGACAGUAUUCU 803 AM07656-SS(NAG37)s(invAb)sgcCfuAfaAfaGfgGfacaguauucus(invAb) 569GCCUAAAAGGGACAGUAUUCU 803 AM07657-SS(NAG37)s(invAb)sgcCfuAfaAfaGfGfIfacaguauucus(invAb) 570GCCUAAAAGGIACAGUAUUCU 818 AM07658-SS(NAG37)s(invAb)sgcCfuAfaAfaGfgIfacaguauucus(invAb) 571GCCUAAAAGGIACAGUAUUCU 818 AM07748-SS(NAG37)s(invAb)sacgggacaGfUfAfuucucaguias(invAb) 572ACGGGACAGUAUUCUCAGUIA 18 AM07749-SS(NAG37)s(invAb)scagggacaGfUfAfuucucagugas(invAb) 573CAGGGACAGUAUUCUCAGUGA 825 AM07751-SS(NAG37)s(invAb)scagggacaGfUfAfuucucaguias(invAb) 574CAGGGACAGUAUUCUCAGUIA 837 AM07752-SS(NAG37)s(invAb)saggggacaGfUfAfuucucagugas(invAb) 575AGGGGACAGUAUUCUCAGUGA 826 AM07754-SS(NAG37)s(invAb)saggggacaGfUfAfuucucaguias(invAb) 576AGGGGACAGUAUUCUCAGUIA 827 AM07759-SS(NAG37)s(invAb)sagaccgCfCfAfaggaugcacuuus(invAb) 577AGACCGCCAAGGAUGCACUUU 828 AM07761-SS(NAG37)s(invAb)sagaccgCfCfAfaggauicacuuus(invAb) 578AGACCGCCAAGGAUICACUUU 829 AM07763-SS(NAG37)s(invAb)sucaguuccCfUfGfaaagacuacus(invAb) 579UCAGUUCCCUGAAAGACUACU 830 AM07766-SS(NAG37)s(invAb)sucaguuccCfUfGfaaagacuacas(invAb) 580UCAGUUCCCUGAAAGACUACA 831 AM07768-SS(NAG37)s(invAb)sacuacuggAfGfCfacciuuaagas(invAb) 581ACUACUGGAGCACCIUUAAGA 832 AM07770-SS(NAG37)s(invAb)sccuguaggUfUfGfcuuaaaaggas(invAb) 582CCUGUAGGUUGCUUAAAAGGA 833 AM07772-SS(NAG37)s(invAb)sgcuguaggUfUfGfcuuaaaaggas(invAb) 583GCUGUAGGUUGCUUAAAAGGA 834 AM07774-SS(NAG37)s(invAb)sggucaguuCfUfCfugaaagacuas(invAb) 584GGUCAGUUCUCUGAAAGACUA 835 (A^(2N)) = 2-aminoadenine nucleotide

The APOC3 RNAi agents described herein are formed by annealing anantisense strand with a sense strand. A sense strand containing asequence listed in Table 2, Table 3, or Table 5 can be hybridized to anyantisense strand containing a sequence listed in Table 2, Table 3, orTable 4, provided the two sequences have a region of at least 85%complementarity over a contiguous 16, 17, 18, 19, 20, or 21 nucleotidesequence.

In some embodiments, the antisense strand of an APOC3 RNAi agentdisclosed herein differs by 0, 1, 2, or 3 nucleotides from any of theantisense strand sequences in Table 4. In some embodiments, the sensestrand of an APOC3 RNAi agent disclosed herein differs by 0, 1, 2, or 3nucleotides from any of the sense strand sequences in Table 5.

In some embodiments, an APOC3 RNAi agent antisense strand comprises anucleotide sequence of any of the sequences in Table 2, Table 3, orTable 4. In some embodiments, an APOC3 RNAi agent antisense strandcomprises the sequence of nucleotides (from 5′ end→3′ end) 1-17, 2-17,1-18, 2-18, 1-19, 2-19, 1-20, 2-20, 1-21, 2-21, 1-22, 2-22, 1-23, 2-23,1-24, or 2-24, 1-25, 2-25, 1-16, or 2-16 of any of the sequences inTable 2, Table 3, or Table 4. In certain embodiments, an APOC3 RNAiagent antisense strand comprises or consists of a modified sequence ofany one of the modified sequences in Table 4. In certain embodiments, anAPOC3 RNAi agent antisense strand comprises or consists of a modifiedsequence of any one of the modified sequences in Table 3.

In some embodiments, an APOC3 RNAi agent sense strand comprises thenucleotide sequence of any of the sequences in Table 2, Table 3, orTable 5. In some embodiments, an APOC3 RNAi agent sense strand comprisesthe sequence of nucleotides (from 5′ end→3′ end) 1-17, 2-17, 3-17, 4-17,1-18, 2-18, 3-18, 4-18, 1-19, 2-19, 3-19, 4-19, 1-20, 2-20, 3-20, 4-20,1-21, 2-21, 3-21, 4-21, 1-22, 2-22, 3-22, 4-22, 1-23, 2-23, 3-23, 4-23,1-24, 2-24, 3-24, 4-24, 1-25, 2-25, 3-25, 4-25, 1-26, 2-26, 3-26, or4-26, of any of the sequences in Table 2, Table 3, or Table 5. Incertain embodiments, an APOC3 RNAi agent sense strand comprises orconsists of a modified sequence of any one of the modified sequences inTable 5. In certain embodiments, an APOC3 RNAi agent sense strandcomprises or consists of a modified sequence of any one of the modifiedsequences in Table 3.

For the APOC3 RNAi agents disclosed herein, the nucleotide at position 1of the antisense strand (from 5′ end→3′ end) can be perfectlycomplementary to an APOC3 gene, or can be non-complementary to an APOC3gene. In some embodiments, the nucleotide at position 1 of the antisensestrand (from 5′ end→3′ end) is a U, A, or dT (or a modified versionthereof). In some embodiments, the nucleotide at position 1 of theantisense strand (from 5′ end→3′ end) forms an A:U or U:A base pair withthe sense strand.

In some embodiments, an APOC3 RNAi agent antisense strand comprises thesequence of nucleotides (from 5′ end→3′ end) 2-18 or 2-19 of any of theantisense strand sequences in Table 2 or Table 4. In some embodiments,an APOC3 RNAi sense strand comprises the sequence of nucleotides (from5′ end→3′ end) 1-17 or 1-18 of any of the sense strand sequences inTable 2 or Table 5.

In some embodiments, an APOC3 RNAi agent includes (i) an antisensestrand comprising the sequence of nucleotides (from 5′ end→3′ end) 2-18or 2-19 of any of the antisense strand sequences in Table 2, Table 3, orTable 4, and (ii) a sense strand comprising the sequence of nucleotides(from 5′ end→3′ end) 1-17 or 1-18 of any of the sense strand sequencesin Table 2, Table 3, or Table 5.

A sense strand containing a sequence listed in Table 2. Table 3, orTable 5 can be hybridized to any antisense strand containing a sequencelisted in Table 2, Table 3, or Table 4, provided the two sequences havea region of at least 85% complementarity over a contiguous 16, 17, 18,19, 20, or 21 nucleotide sequence. In some embodiments, the APOC3 RNAiagent has a sense strand consisting of the modified sequence of any ofthe modified sequences in Table 5, and an antisense strand consisting ofthe modified sequence of any of the modified sequences in Table 4.Representative sequence pairings are exemplified by the Duplex ID Nos.shown in Table 3 and Table 6.

In some embodiments, an APOC3 RNAi agent comprises any of the duplexesrepresented by any of the Duplex ID Nos. presented herein. In someembodiments, an APOC3 RNAi agent consists of any of the duplexesrepresented by any of the Duplex ID Nos. presented herein. In someembodiments, an APOC3 RNAi agent comprises the sense strand andantisense strand nucleotide sequences of any of the duplexes representedby any of the Duplex ID Nos. presented herein. In some embodiments, anAPOC3 RNAi agent includes the sense strand and antisense strandnucleotide sequences of any of the duplexes represented by any of theDuplex ID Nos. presented herein and a targeting group and/or linkinggroup, wherein the targeting group and/or linking group is covalentlylinked (i.e., conjugated) to the sense strand or the antisense strand.In some embodiments, an APOC3 RNAi agent includes the sense strand andantisense strand modified nucleotide sequences of any of the duplexesrepresented by any of the Duplex ID Nos. presented herein. In someembodiments, an APOC3 RNAi agent comprises the sense strand andantisense strand modified nucleotide sequences of any of the duplexesrepresented by any of the Duplex ID Nos. presented herein and atargeting group and/or linking group, wherein the targeting group and/orlinking group is covalently linked to the sense strand or the antisensestrand.

In some embodiments, an APOC3 RNAi agent comprises an antisense strandand a sense strand having the nucleotide sequences of any of theantisense strand/sense strand duplexes of Table 2, Table 3, or Table 6,and comprises an asialoglycoprotein receptor ligand targeting group.

In some embodiments, an APOC3 RNAi agent comprises an antisense strandand a sense strand having the nucleotide sequences of any of theantisense strand/sense strand duplexes of Table 2, Table 3, or Table 6,and further comprises a targeting group selected from the groupconsisting of (NAG13), (NAG13)s, (NAG18), (NAG18)s, (NAG24), (NAG24)s,(NAG25), (NAG25)s, (NAG26), (NAG26)s, (NAG27), (NAG27)s, (NAG28),(NAG28)s, (NAG29), (NAG29)s, (NAG30), (NAG30)s, (NAG31), (NAG31)s,(NAG32), (NAG32)s, (NAG33), (NAG33)s, (NAG34), (NAG34)s, (NAG35),(NAG35)s, (NAG36), (NAG36)s, (NAG37), (NAG37)s, (NAG38), (NAG38)s,(NAG39), (NAG39)s. In some embodiments, the targeting group is (NAG25)or (NAG25)s as defined in Table 7. In other embodiments, the targetinggroup is (NAG37) or (NAG37)s as defined in Table 7.

In some embodiments, an APOC3 RNAi agent comprises an antisense strandand a sense strand having the modified nucleotide sequence of any of theantisense strand and/or sense strand nucleotide sequences of any of theduplexes of Table 2, Table 3, or Table 6.

In some embodiments, an APOC3 RNAi agent comprises an antisense strandand a sense strand having a modified nucleotide sequence of any of theantisense strand and/or sense strand nucleotide sequences of any of theduplexes of Table 2, Table 3, or Table 6, and comprises anasialoglycoprotein receptor ligand targeting group.

In some embodiments, an APOC3 RNAi agent comprises any of the duplexesof Table 2, Table 3, or Table 6.

In some embodiments, an APOC3 RNAi agent consists of any of the duplexesof Table 2, Table 3, or Table 6.

TABLE 6 APOC3 RNAi Agents Identified by Duplex ID NO. with CorrespondingSense and Antisense Strands. Duplex Antisense Sense ID Strand ID StrandID AD04812 AM06203-AS AM06206-SS AD04813 AM06204-AS AM06207-SS AD04814AM06203-AS AM06208-SS AD04815 AM06205-AS AM06209-SS AD04816 AM06210-ASAM06212-SS AD04817 AM06211-AS AM06213-SS AD04818 AM06214-AS AM06216-SSAD04819 AM06215-AS AM06217-SS AD04820 AM06218-AS AM06220-SS AD04821AM06219-AS AM06221-SS AD04860 AM06262-AS AM06264-SS AD04861 AM06263-ASAM06265-SS AD04862 AM06266-AS AM06268-SS AD04863 AM06266-AS AM06269-SSAD04864 AM06267-AS AM06270-SS AD04865 AM06266-AS AM06271-SS AD04866AM06272-AS AM06274-SS AD04867 AM06273-AS AM06275-SS AD04868 AM06276-ASAM06278-SS AD04869 AM06277-AS AM06279-SS AD04870 AM06277-AS AM06280-SSAD04886 AM06309-AS AM06311-SS AD04887 AM06310-AS AM06312-SS AD04888AM06309-AS AM06313-SS AD04889 AM06314-AS AM06316-SS AD04890 AM06315-ASAM06317-SS AD04891 AM06318-AS AM06321-SS AD04892 AM06319-AS AM06322-SSAD04893 AM06320-AS AM06323-SS AD04894 AM06324-AS AM06326-SS AD04895AM06325-AS AM06327-SS AD04896 AM06328-AS AM06329-SS AD04897 AM06330-ASAM06332-SS AD04898 AM06331-AS AM06333-SS AD04987 AM06469-AS AM06206-SSAD04988 AM06469-AS AM06208-SS AD04989 AM06471-AS AM06470-SS AD04990AM06205-AS AM06470-SS AD04991 AM06472-AS AM06208-SS AD04992 AM06204-ASAM06473-SS AD04993 AM06475-AS AM06474-SS AD04994 AM06476-AS AM06207-SSAD04995 AM06477-AS AM06207-SS AD04996 AM06478-AS AM06209-SS AD04997AM06471-AS AM06479-SS AD04998 AM06481-AS AM06480-SS AD05007 AM06507-ASAM06506-SS AD05008 AM06509-AS AM06508-SS AD05009 AM06511-AS AM06510-SSAD05010 AM06513-AS AM06512-SS AD05011 AM06514-AS AM06326-SS AD05012AM06324-AS AM06515-SS AD05013 AM06517-AS AM06516-SS AD05014 AM06518-ASAM06326-SS AD05015 AM06519-AS AM06326-SS AD05016 AM06521-AS AM06520-SSAD05017 AM06523-AS AM06522-SS AD05127 AM06712-AS AM06711-SS AD05128AM06714-AS AM06713-SS AD05129 AM06716-AS AM06715-SS AD05130 AM06718-ASAM06717-SS AD05131 AM06720-AS AM06719-SS AD05132 AM06722-AS AM06721-SSAD05133 AM06724-AS AM06723-SS AD05134 AM06726-AS AM06725-SS AD05135AM06728-AS AM06727-SS AD05136 AM06730-AS AM06729-SS AD05137 AM06732-ASAM06731-SS AD05138 AM06734-AS AM06733-SS AD05139 AM06736-AS AM06735-SSAD05140 AM06738-AS AM06737-SS AD05141 AM06740-AS AM06739-SS AD05142AM06741-AS AM06739-SS AD05143 AM06743-AS AM06742-SS AD05144 AM06745-ASAM06744-SS AD05167 AM06780-AS AM06779-SS AD05168 AM06780-AS AM06781-SSAD05169 AM06783-AS AM06782-SS AD05170 AM06784-AS AM06735-SS AD05171AM06786-AS AM06785-SS AD05172 AM06786-AS AM06787-SS AD05173 AM06716-ASAM06788-SS AD05174 AM06716-AS AM06789-SS AD05175 AM06716-AS AM06790-SSAD05176 AM06783-AS AM06791-SS AD05177 AM06784-AS AM06792-SS AD05215AM06862-AS AM06779-SS AD05216 AM06780-AS AM06863-SS AD05217 AM06865-ASAM06864-SS AD05218 AM06507-AS AM06866-SS AD05219 AM06868-AS AM06867-SSAD05220 AM06870-AS AM06869-SS AD05221 AM06872-AS AM06871-SS AD05222AM06874-AS AM06873-SS AD05223 AM06876-AS AM06875-SS AD05239 AM06908-ASAM06907-SS AD05249 AM06741-AS AM06922-SS AD05250 AM06783-AS AM06923-SSAD05251 AM06783-AS AM06924-SS AD05252 AM06743-AS AM06925-SS AD05253AM06908-AS AM06926-SS AD05254 AM06928-AS AM06927-SS AD05255 AM06786-ASAM06929-SS AD05258 AM06786-AS AM06932-SS AD05259 AM06786-AS AM06933-SSAD05260 AM06517-AS AM06934-SS AD05275 AM06872-AS AM06948-SS AD05276AM06872-AS AM06949-SS AD05277 AM06951-AS AM06950-SS AD05278 AM06953-ASAM06952-SS AD05279 AM06951-AS AM06954-SS AD05280 AM06956-AS AM06955-SSAD05281 AM06958-AS AM06957-SS AD05282 AM06959-AS AM06875-SS AD05283AM06961-AS AM06960-SS AD05284 AM06963-AS AM06962-SS AD05285 AM06876-ASAM06964-SS AD05286 AM06876-AS AM06965-SS AD05287 AM06876-AS AM06966-SSAD05299 AM06988-AS AM06987-SS AD05431 AM06517-AS AM07178-SS AD05432AM07179-AS AM06934-SS AD05433 AM06517-AS AM07180-SS AD05434 AM06872-ASAM07181-SS AD05435 AM07182-AS AM06871-SS AD05436 AM06872-AS AM07183-SSAD05437 AM06876-AS AM07184-SS AD05438 AM07185-AS AM06875-SS AD05439AM06876-AS AM07186-SS AD05440 AM06786-AS AM07187-SS AD05441 AM07188-ASAM06785-SS AD05442 AM06786-AS AM07189-SS AD05443 AM07190-AS AM06924-SSAD05444 AM06783-AS AM07191-SS AD05445 AM06870-AS AM07192-SS AD05446AM07193-AS AM06869-SS AD05447 AM06870-AS AM07194-SS AD05535 AM06517-ASAM07309-SS AD05536 AM06517-AS AM07310-SS AD05537 AM06872-AS AM07311-SSAD05538 AM06872-AS AM07312-SS AD05539 AM06872-AS AM07313-SS AD05540AM06876-AS AM07314-SS AD05541 AM06876-AS AM07315-SS AD05542 AM06876-ASAM07316-SS AD05543 AM06786-AS AM07317-SS AD05544 AM06786-AS AM07318-SSAD05545 AM06783-AS AM07319-SS AD05546 AM06870-AS AM07320-SS AD05547AM06870-AS AM07321-SS AD05705 AM06961-AS AM07515-SS AD05706 AM06961-ASAM07516-SS AD05707 AM07518-AS AM07517-SS AD05708 AM07520-AS AM07519-SSAD05709 AM07522-AS AM07521-SS AD05710 AM07190-AS AM06782-SS AD05711AM06783-AS AM07523-SS AD05712 AM07524-AS AM06924-SS AD05713 AM06908-ASAM07525-SS AD05714 AM06870-AS AM07526-SS AD05761 AM06961-AS AM07598-SSAD05762 AM06961-AS AM07599-SS AD05763 AM07600-AS AM06960-SS AD05764AM07600-AS AM07516-SS AD05765 AM07600-AS AM07598-SS AD05766 AM07600-ASAM07599-SS AD05767 AM07524-AS AM06782-SS AD05768 AM07524-AS AM07601-SSAD05769 AM07524-AS AM07602-SS AD05811 AM06870-AS AM07644-SS AD05812AM07645-AS AM07644-SS AD05813 AM07645-AS AM07646-SS AD05814 AM07645-ASAM07647-SS AD05815 AM06870-AS AM07648-SS AD05816 AM06870-AS AM07649-SSAD05817 AM07645-AS AM07650-SS AD05818 AM07645-AS AM07651-SS AD05819AM06870-AS AM07652-SS AD05820 AM07645-AS AM07653-SS AD05821 AM07645-ASAM07654-SS AD05822 AM07600-AS AM07655-SS AD05823 AM07600-AS AM07656-SSAD05824 AM07600-AS AM07657-SS AD05825 AM07600-AS AM07658-SS AD05876AM06743-AS AM07748-SS AD05877 AM07750-AS AM07749-SS AD05878 AM07750-ASAM07751-SS AD05879 AM07753-AS AM07752-SS AD05880 AM07753-AS AM07754-SSAD05881 AM07755-AS AM06782-SS AD05882 AM07756-AS AM06782-SS AD05883AM07757-AS AM06782-SS AD05884 AM07758-AS AM06782-SS AD05885 AM07755-ASAM06924-SS AD05886 AM07756-AS AM06924-SS AD05887 AM07757-AS AM06924-SSAD05888 AM07758-AS AM06924-SS AD05889 AM07760-AS AM07759-SS AD05890AM07762-AS AM07761-SS AD05891 AM07764-AS AM07763-SS AD05892 AM07765-ASAM07763-SS AD05893 AM07767-AS AM07766-SS AD05894 AM07769-AS AM07768-SSAD05895 AM07771-AS AM07770-SS AD05896 AM07773-AS AM07772-SS AD05897AM07775-AS AM07774-SS

In some embodiments, an APOC3 RNAi agent is prepared or provided as asalt, mixed salt, or a free-acid. The RNAi agents described herein, upondelivery to a cell expressing an APOC3 gene, inhibit or knockdownexpression of one or more APOC3 genes in vivo.

Targeting Groups, Linking Groups, and Delivery Vehicles

In some embodiments, an APOC3 RNAi agent is conjugated to one or morenon-nucleotide groups including, but not limited to a targeting group,linking group, delivery polymer, or a delivery vehicle. Thenon-nucleotide group can enhance targeting, delivery or attachment ofthe RNAi agent. Examples of targeting groups and linking groups areprovided in Table 7. The non-nucleotide group can be covalently linkedto the 3′ and/or 5′ end of either the sense strand and/or the antisensestrand. In some embodiments, an APOC3 RNAi agent contains anon-nucleotide group linked to the 3′ and/or 5′ end of the sense strand.In some embodiments, a non-nucleotide group is linked to the 5′ end ofan APOC3 RNAi agent sense strand. A non-nucleotide group may be linkeddirectly or indirectly to the RNAi agent via a linker/linking group. Insome embodiments, a non-nucleotide group is linked to the RNAi agent viaa labile, cleavable, or reversible bond or linker.

In some embodiments, a non-nucleotide group enhances the pharmacokineticor biodistribution properties of an RNAi agent or conjugate to which itis attached to improve cell- or tissue-specific distribution andcell-specific uptake of the RNAi agent or conjugate. In someembodiments, a non-nucleotide group enhances endocytosis of the RNAiagent.

Targeting groups or targeting moieties can enhance the pharmacokineticor biodistribution properties of a conjugate or RNAi agent to which theyare attached to improve cell-specific distribution and cell-specificuptake of the conjugate or RNAi agent. A targeting group can bemonovalent, divalent, trivalent, tetravalent, or have higher valency forthe target to which it is directed. Representative targeting groupsinclude, without limitation, compounds with affinity to cell surfacemolecules, cell receptor ligands, haptens, antibodies, monoclonalantibodies, antibody fragments, and antibody mimics with affinity tocell surface molecules. In some embodiments, a targeting group is linkedto an RNAi agent using a linker, such as a PEG linker or one, two, orthree abasic and/or ribitol (abasic ribose) residues, which in someinstances can serve as linkers. In some embodiments, a targeting groupcomprises a galactose-derivative cluster.

The APOC3 RNAi agents described herein can be synthesized having areactive group, such as an amine group, at the 5′-terminus. The reactivegroup can be used to subsequently attach a targeting group using methodstypical in the art.

In some embodiments, a targeting group comprises an asialoglycoproteinreceptor ligand. As used herein, an asialoglycoprotein receptor ligandis a ligand that contains a compound having affinity for theasialoglycoprotein receptor, which is highly expressed on hepatocytes.In some embodiments, an asialoglycoprotein receptor ligand includes orconsists of one or more galactose derivatives. As used herein, the termgalactose derivative includes both galactose and derivatives ofgalactose having affinity for the asialoglycoprotein receptor that isequal to or greater than that of galactose. Galactose derivativesinclude, but are not limited to: galactose, galactosamine,N-formylgalactosamine, N-acetyl-galactosamine,N-propionyl-galactosamine, N-n-butanoyl-galactosamine, andN-iso-butanoylgalactos-amine (see for example: S. T. Iobst and K.Drickamer, J. B. C., 1996, 271, 6686). Galactose derivatives, andclusters of galactose derivatives, that are useful for in vivo targetingof oligonucleotides and other molecules to the liver are known in theart (see, for example, Baenziger and Fiete, 1980, Cell, 22, 611-620;Connolly et al., 1982, J. Biol. Chem., 257, 939-945).

Galactose derivatives have been used to target molecules to hepatocytesin vivo through their binding to the asialoglycoprotein receptorexpressed on the surface of hepatocytes. Binding of asialoglycoproteinreceptor ligands to the asialoglycoprotein receptor(s) facilitatescell-specific targeting to hepatocytes and endocytosis of the moleculeinto hepatocytes. Asialoglycoprotein receptor ligands can be monomeric(e.g., having a single galactose derivative) or multimeric (e.g., havingmultiple galactose derivatives). The galactose derivative or galactosederivative cluster can be attached to the 3′ or 5′ end of the sense orantisense strand of the RNAi agent using methods known in the art. Thepreparation of targeting groups, such as galactose derivative clusters,is described in, for example, International Patent ApplicationPublication No. WO 2018/044350 to Arrowhead Pharmaceuticals, Inc., andInternational Patent Application Publication No. WO 2017/156012 toArrowhead Pharmaceuticals, Inc., the contents of both of which areincorporated by reference herein in their entirety.

As used herein, a galactose derivative cluster comprises a moleculehaving two to four terminal galactose derivatives. A terminal galactosederivative is attached to a molecule through its C-1 carbon. In someembodiments, the galactose derivative cluster is a galactose derivativetrimer (also referred to as tri-antennary galactose derivative ortri-valent galactose derivative). In some embodiments, the galactosederivative cluster comprises N-acetyl-galactosamines. In someembodiments, the galactose derivative cluster comprises threeN-acetyl-galactosamines. In some embodiments, the galactose derivativecluster is a galactose derivative tetramer (also referred to astetra-antennary galactose derivative or tetra-valent galactosederivative). In some embodiments, the galactose derivative clustercomprises four N-acetyl-galactosamines.

As used herein, a galactose derivative trimer contains three galactosederivatives, each linked to a central branch point. As used herein, agalactose derivative tetramer contains four galactose derivatives, eachlinked to a central branch point. The galactose derivatives can beattached to the central branch point through the C-1 carbons of thesaccharides. In some embodiments, the galactose derivatives are linkedto the branch point via linkers or spacers. In some embodiments, thelinker or spacer is a flexible hydrophilic spacer, such as a PEG group(see, for example, U.S. Pat. No. 5,885,968; Biessen et al. J. Med. Chem.1995 Vol. 39 p. 1538-1546). In some embodiments, the PEG spacer is aPEG3 spacer. The branch point can be any small molecule which permitsattachment of three galactose derivatives and further permits attachmentof the branch point to the RNAi agent. An example of branch point groupis a di-lysine or di-glutamate. Attachment of the branch point to theRNAi agent can occur through a linker or spacer. In some embodiments,the linker or spacer comprises a flexible hydrophilic spacer, such as,but not limited to, a PEG spacer. In some embodiments, the linkercomprises a rigid linker, such as a cyclic group. In some embodiments, agalactose derivative comprises or consists of N-acetyl-galactosamine. Insome embodiments, the galactose derivative cluster is comprised of agalactose derivative tetramer, which can be, for example, anN-acetyl-galactosamine tetramer.

Embodiments of the present disclosure include pharmaceuticalcompositions for delivering an APOC3 RNAi agent to a liver cell in vivo.Such pharmaceutical compositions can include, for example, an APOC3 RNAiagent conjugated to a galactose derivative cluster.

In some embodiments, the galactose derivative cluster is comprised of agalactose derivative trimer, which can be, for example, anN-acetyl-galactosamine trimer, or galactose derivative tetramer, whichcan be, for example, an N-acetyl-galactosamine tetramer.

Targeting groups include, but are not limited to, (PAZ), (NAG13),(NAG13)s, (NAG18), (NAG18)s, (NAG24), (NAG24)s, (NAG25), (NAG25)s,(NAG26), (NAG26)s, (NAG27), (NAG27)s, (NAG28), (NAG28)s, (NAG29),(NAG29)s, (NAG30), (NAG30)s, (NAG31), (NAG31)s, (NAG32), (NAG32)s,(NAG33), (NAG33)s, (NAG34), (NAG34)s, (NAG35), (NAG35)s, (NAG36),(NAG36)s, (NAG37), (NAG37)s, (NAG38), (NAG38)s, (NAG39), and (NAG39)s asdefined in Table 7. Other targeting groups, including galactose clustertargeting ligands, are known in the art.

In some embodiments, a linking group is conjugated to the RNAi agent.The linking group facilitates covalent linkage of the agent to atargeting group or delivery polymer or delivery vehicle. The linkinggroup can be linked to the 3′ or the 5′ end of the RNAi agent sensestrand or antisense strand. In some embodiments, the linking group islinked to the RNAi agent sense strand. In some embodiments, the linkinggroup is conjugated to the 5′ or 3′ end of an RNAi agent sense strand.In some embodiments, a linking group is conjugated to the 5′ end of anRNAi agent sense strand. Examples of linking groups, can include, butare not limited to: reactive groups such a primary amines and alkynes,alkyl groups, abasic nucleotides, ribitol (abasic ribose), and/or PEGgroups.

A linker or linking group is a connection between two atoms that linksone chemical group (such as an RNAi agent) or segment of interest toanother chemical group (such as a targeting group or delivery polymer)or segment of interest via one or more covalent bonds. A labile linkagecontains a labile bond. A linkage may optionally include a spacer thatincreases the distance between the two joined atoms. A spacer canfurther add flexibility and/or length to the linkage. Spacers caninclude, but are not be limited to, alkyl groups, alkenyl groups,alkynyl groups, aryl groups, aralkyl groups, aralkenyl groups, andaralkynyl groups; each of which can contain one or more heteroatoms,heterocycles, amino acids, nucleotides, and saccharides. Spacer groupsare well known in the art and the preceding list is not meant to limitthe scope of the description.

Any of the APOC3 RNAi agent nucleotide sequences listed in Tables 2, 3,4, or 5, whether modified or unmodified, may contain 3′ or 5′ targetinggroup or linking group. Any of the APOC3 RNAi agent sequences listed inTable 4 or 5 which contain a 3′ or 5′ targeting group or linking group,may alternatively contain no 3′ or 5′ targeting group or linking group,or may contain a different 3′ or 5′ targeting group or linking groupincluding, but not limited to, those depicted in Table 7. Any of theAPOC3 RNAi agent duplexes listed in Table 2, Table 3, or Table 6,whether modified or unmodified, may further comprise a targeting groupor linking group, including, but not limited to, those depicted in Table7, and the targeting group or linking group may be attached to the 3′ or5′ terminus of either the sense strand or the antisense strand of theAPOC3 RNAi agent duplex.

Examples of targeting groups and linking groups are provided in Table 7.Table 5 provides several embodiments of APOC3 RNAi agent sense strandshaving a targeting group or linking group linked to the 5′ or 3′ end.

TABLE 7 Structures Representing Various Modified Nucleotides, TargetingGroups, and Linking Groups.

When positioned internally in oligonucleotide:

When positioned internally in oligonucleotide:

When positioned at the 3′ terminal end of oligonucleotide:

In each of the above structures in Table 7, NAG comprises anN-acetyl-galactosamine or another galactose derivative, as would beunderstood by a person of ordinary skill in the art to be attached inview of the structures above and description provided herein. Forexample, in some embodiments, NAG in the structures provided in Table 7is represented by the following structure:

Each (NAGx) may be attached to an APOC3 RNAi agent via a phosphate group(as in (NAG25), (NAG30), and (NAG31)), or a phosphorothioate group, (asis (NAG25)s, (NAG29)s, (NAG30)s, (NAG31)s, or (NAG37)s), or anotherlinking group.

Other linking groups known in the art may be used.

In some embodiments, a delivery vehicle can be used to deliver an RNAiagent to a cell or tissue. A delivery vehicle is a compound thatimproves delivery of the RNAi agent to a cell or tissue. A deliveryvehicle can include, or consist of, but is not limited to: a polymer,such as an amphipathic polymer, a membrane active polymer, a peptide, amelittin peptide, a melittin-like peptide (MLP), a lipid, a reversiblymodified polymer or peptide, or a reversibly modified membrane activepolyamine. In some embodiments, the RNAi agents can be combined withlipids, nanoparticles, polymers, liposomes, micelles, DPCs or otherdelivery systems available in the art. The RNAi agents can also bechemically conjugated to targeting groups, lipids (including, but notlimited to cholesterol and cholesteryl derivatives), nanoparticles,polymers, liposomes, micelles, DPCs (see, for example WO 2000/053722, WO2008/0022309, WO 2011/104169, and WO 2012/083185, WO 2013/032829, WO2013/158141, each of which is incorporated herein by reference), orother delivery systems available in the art.

Pharmaceutical Compositions and Formulations

The APOC3 RNAi agents disclosed herein can be prepared as pharmaceuticalcompositions or formulations. In some embodiments, pharmaceuticalcompositions include at least one APOC3 RNAi agent. These pharmaceuticalcompositions are particularly useful in the inhibition of the expressionof the target mRNA in a target cell, a group of cells, a tissue, or anorganism. The pharmaceutical compositions can be used to treat a subjecthaving a disease or disorder that would benefit from reduction in thelevel of the target mRNA, or inhibition in expression of the targetgene. The pharmaceutical compositions can be used to treat a subject atrisk of developing a disease, disorder, or condition that would benefitfrom reduction of the level of the target mRNA or an inhibition inexpression the target gene. In one embodiment, the method includesadministering an APOC3 RNAi agent linked to a targeting ligand asdescribed herein, to a subject to be treated. In some embodiments, oneor more pharmaceutically acceptable excipients (including vehicles,carriers, diluents, and/or delivery polymers) are added to thepharmaceutical compositions including an APOC3 RNAi agent, therebyforming a pharmaceutical formulation suitable for in vivo delivery to asubject, including a human.

The pharmaceutical compositions that include an APOC3 RNAi agent andmethods disclosed herein may decrease the level of the target mRNA in acell, group of cells, group of cells, tissue, or subject, including:administering to the subject a therapeutically effective amount of aherein described APOC3 RNAi agent, thereby inhibiting the expression ofAPOC3 mRNA in the subject. In some embodiments, the subject has beenpreviously identified as having a pathogenic upregulation of the targetgene in the targeted cell or tissue.

In some embodiments, the described pharmaceutical compositions includingan APOC3 RNAi agent are used for treating or managing clinicalpresentations associated with elevated TG levels and/or over-expressionof APOC3 mRNA in a subject. In some embodiments, a therapeutically(including prophylactically) effective amount of one or more ofpharmaceutical compositions is administered to a subject in need of suchtreatment (including the prevention or management of symptoms, diseases,or disorders). In some embodiments, administration of any of thedisclosed APOC3 RNAi agents can be used to decrease the number,severity, and/or frequency of symptoms of a disease in a subject.

The described pharmaceutical compositions including an APOC3 RNAi agentcan be used to treat at least one symptom in a subject having a diseaseor disorder that would benefit from reduction or inhibition inexpression of APOC3 mRNA. In some embodiments, the subject isadministered a therapeutically effective amount of one or morepharmaceutical compositions including an APOC3 RNAi agent therebytreating the symptom. In other embodiments, the subject is administereda prophylactically effective amount of one or more APOC3 RNAi agents,thereby preventing the at least one symptom.

The route of administration is the path by which an APOC3 RNAi agent isbrought into contact with the body. In general, methods of administeringdrugs and oligonucleotides and nucleic acids for treatment of a mammalare well known in the art and can be applied to administration of thecompositions described herein. The APOC3 RNAi agents disclosed hereincan be administered via any suitable route in a preparationappropriately tailored to the particular route. Thus, herein describedpharmaceutical compositions can be administered by injection, forexample, intravenously, intramuscularly, intracutaneously,subcutaneously, intraarticularly, or intraperitoneally. In someembodiments, the herein described pharmaceutical compositions areadministered via subcutaneous injection.

The pharmaceutical compositions including an APOC3 RNAi agent describedherein can be delivered to a cell, group of cells, tissue, or subjectusing oligonucleotide delivery technologies known in the art. Ingeneral, any suitable method recognized in the art for delivering anucleic acid molecule (in vitro or in vivo) can be adapted for use withthe compositions described herein. For example, delivery can be by localadministration, (e.g., direct injection, implantation, or topicaladministering), systemic administration, or subcutaneous, intravenous,intraperitoneal, or parenteral routes, including intracranial (e.g.,intraventricular, intraparenchymal and intrathecal), intramuscular,transdermal, airway (aerosol), nasal, oral, rectal, or topical(including buccal and sublingual) administration. In certainembodiments, the compositions are administered by subcutaneous orintravenous infusion or injection.

Accordingly, in some embodiments, the pharmaceutical compositionsdescribed herein comprise one or more pharmaceutically acceptableexcipients. The pharmaceutical compositions described herein areformulated for administration to a subject.

As used herein, a pharmaceutical composition or medicament includes apharmacologically effective amount of at least one of the describedtherapeutic compounds and one or more pharmaceutically acceptableexcipients. Pharmaceutically acceptable excipients (excipients) aresubstances other than the Active Pharmaceutical Ingredient (API,therapeutic product, e.g., APOC3 RNAi agent) that are intentionallyincluded in the drug delivery system. Excipients do not exert or are notintended to exert a therapeutic effect at the intended dosage.Excipients may act to a) aid in processing of the drug delivery systemduring manufacture, b) protect, support or enhance stability,bioavailability or patient acceptability of the API, c) assist inproduct identification, and/or d) enhance any other attribute of theoverall safety, effectiveness, of delivery of the API during storage oruse. A pharmaceutically acceptable excipient may or may not be an inertsubstance.

Excipients include, but are not limited to: absorption enhancers,anti-adherents, anti-foaming agents, anti-oxidants, binders, bufferingagents, carriers, coating agents, colors, delivery enhancers, deliverypolymers, dextran, dextrose, diluents, disintegrants, emulsifiers,extenders, fillers, flavors, glidants, humectants, lubricants, oils,polymers, preservatives, saline, salts, solvents, sugars, suspendingagents, sustained release matrices, sweeteners, thickening agents,tonicity agents, vehicles, water-repelling agents, and wetting agents.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, Cremophor®ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).Suitable carriers should be stable under the conditions of manufactureand storage and should be preserved against the contaminating action ofmicroorganisms such as bacteria and fungi. The carrier can be a solventor dispersion medium containing, for example, water, ethanol, polyol(for example, glycerol, propylene glycol, and liquid polyethyleneglycol), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, and sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent which delays absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfilter sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle, which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation include vacuumdrying and freeze-drying which yields a powder of the active ingredientplus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Formulations suitable for intra-articular administration can be in theform of a sterile aqueous preparation of the drug that can be inmicrocrystalline form, for example, in the form of an aqueousmicrocrystalline suspension. Liposomal formulations or biodegradablepolymer systems can also be used to present the drug for bothintra-articular and ophthalmic administration.

The active compounds can be prepared with carriers that will protect thecompound against rapid elimination from the body, such as a controlledrelease formulation, including implants and microencapsulated deliverysystems. Biodegradable, biocompatible polymers can be used, such asethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Methods for preparation of suchformulations will be apparent to those skilled in the art. Liposomalsuspensions can also be used as pharmaceutically acceptable carriers.These can be prepared according to methods known to those skilled in theart, for example, as described in U.S. Pat. No. 4,522,811.

The APOC3 RNAi agents can be formulated in compositions in dosage unitform for ease of administration and uniformity of dosage. Dosage unitform refers to physically discrete units suited as unitary dosages forthe subject to be treated; each unit containing a predetermined quantityof active compound calculated to produce the desired therapeutic effectin association with the required pharmaceutical carrier. Thespecification for the dosage unit forms of the disclosure are dictatedby and directly dependent on the unique characteristics of the activecompound and the therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

A pharmaceutical composition can contain other additional componentscommonly found in pharmaceutical compositions. Such additionalcomponents include, but are not limited to: anti-pruritics, astringents,local anesthetics, analgesics, antihistamines, or anti-inflammatoryagents (e.g., acetaminophen, NSAIDs, diphenhydramine, etc.). It is alsoenvisioned that cells, tissues or isolated organs that express orcomprise the herein defined RNAi agents may be used as “pharmaceuticalcompositions.” As used herein, “pharmacologically effective amount,”“therapeutically effective amount,” or simply “effective amount” refersto that amount of an RNAi agent to produce a pharmacological,therapeutic or preventive result.

In some embodiments, the methods disclosed herein further comprise thestep of administering a second therapeutic or treatment in addition toadministering an RNAi agent disclosed herein. In some embodiments, thesecond therapeutic is another APOC3 RNAi agent (e.g., an APOC3 RNAiagent which targets a different sequence within the APOC3 target). Inother embodiments, the second therapeutic can be a small molecule drug,an antibody, an antibody fragment. or an aptamer.

Generally, an effective amount of an active compound will be in therange of from about 0.1 to about 100 mg/kg of body weight/day, e.g.,from about 1.0 to about 50 mg/kg of body weight/day. In someembodiments, an effective amount of an active compound will be in therange of from about 0.25 to about 5 mg/kg of body weight per dose. Insome embodiments, an effective amount of an active ingredient will be inthe range of from about 0.5 to about 4 mg/kg of body weight per dose.The amount administered will also likely depend on such variables as theoverall health status of the patient, the relative biological efficacyof the compound delivered, the formulation of the drug, the presence andtypes of excipients in the formulation, and the route of administration.Also, it is to be understood that the initial dosage administered can,in some instances, be increased beyond the above upper level in order torapidly achieve the desired blood-level or tissue level, or the initialdosage can, in some instances, be smaller than the optimum.

For treatment of disease or for formation of a medicament or compositionfor treatment of a disease, the pharmaceutical compositions describedherein including an APOC3 RNAi agent can be combined with an excipientor with a second therapeutic agent or treatment including, but notlimited to: a second or other RNAi agent, a small molecule drug, anantibody, an antibody fragment, peptide and/or a aptamer.

The described APOC3 RNAi agents, when added to pharmaceuticallyacceptable excipients or adjuvants, can be packaged into kits,containers, packs, or dispensers. The pharmaceutical compositionsdescribed herein may be packaged in pre-filled syringes or vials.

Methods of Treatment and Inhibition of Expression

The APOC3 RNAi agents disclosed herein can be used to treat a subject(e.g., a human or other mammal) having a disease or disorder that wouldbenefit from administration of the compound. In some embodiments, theRNAi agents disclosed herein can be used to treat a subject (e.g., ahuman) having a disease or disorder that would benefit from a reductionand/or an inhibition in expression of APOC3 mRNA, for example, a subjectthat has been diagnosed with or is at risk of developing symptomsrelated to obesity, hyperlipidemia, hypertriglyceridemia, abnormal lipidand/or cholesterol metabolism, atherosclerosis, cardiovascular disease,coronary artery disease, hypertriglyceridemia mediated pancreatitis,metabolic syndrome, type II diabetes mellitus, familial chylomicronemniasyndrome, familial partial lipodystrophy, and/or other metabolic-relateddisorders and diseases.

The subject is administered a therapeutically effective amount of anyone or more RNAi agents. The subject can be a human, patient, or humanpatient. The subject may be an adult, adolescent, child, or infant.Administration of a pharmaceutical composition described herein can beto a human being or animal.

In some embodiments, the APOC3 RNAi agents described herein are used totreat a subject with an APOC3-related disease or disorder. In someembodiments, the APOC3 RNAi agents described herein are used to treat asubject that would benefit from a reduction and/or inhibition of APOC3gene expression. In some embodiments, the described APOC3 RNAi agentsare used to treat (including prophylactically) at least one symptom orpathological stated mediated at least in part by APOC3 gene expression.The subject is administered a therapeutically effective amount of anyone or more of the described RNAi agents. In some embodiments, thesubject is administered a prophylactically effective amount of any oneor more of the described RNAi agents, thereby preventing the at leastone symptom.

In certain embodiments, the present invention provides methods fortreatment of diseases, disorders, conditions, or pathological statesmediated at least in part by APOC3 expression, in a patient in needthereof, wherein the methods include administering to the patient any ofthe APOC3 RNAi agents described herein.

In some embodiments, the APOC3 RNAi agents are used to treat or manage aclinical presentation of a subject with an APOC3-related disease ordisorder. The subject is administered a therapeutically effective amountof one or more of the APOC3 RNAi agents or APOC3 RNAi agent-containingcompositions described herein. In some embodiments, the method comprisesadministering a composition comprising an APOC3 RNAi agent describedherein to a subject to be treated.

In some embodiments, the gene expression level and/or mRNA level of anAPOC3 gene in a subject to whom a described APOC3 RNAi agent isadministered is reduced by at least about 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99%, or greater than99% relative to the subject prior to being administered the APOC3 RNAiagent or to a subject not receiving the APOC3 RNAi agent. The geneexpression level and/or mRNA level in the subject is reduced in a cell,group of cells, and/or tissue of the subject.

In some embodiments, the protein level of APOC3 in a subject to whom adescribed APOC3 RNAi agent has been administered is reduced by at leastabout 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, or greater than 99% relative to the subjectprior to being administered the APOC3 RNAi agent or to a subject notreceiving the APOC3 RNAi agent. The protein level in the subject isreduced in a cell, group of cells, tissue, blood, and/or other fluid ofthe subject.

In some embodiments, the triglyceride (TG) levels in a subject to whom adescribed APOC3 RNAi agent has been administered is reduced by at leastabout 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% relative to thesubject prior to being administered the APOC3 RNAi agent or to a subjectnot receiving the APOC3 RNAi agent. The TG level in the subject may bereduced in a cell, group of cells, tissue, blood, and/or other fluid ofthe subject.

In some embodiments, the total cholesterol levels in a subject to whom adescribed APOC3 RNAi agent has been administered is reduced by at leastabout 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% relative to thesubject prior to being administered the APOC3 RNAi agent or to a subjectnot receiving the APOC3 RNAi agent. In some embodiments, the low-densitylipoprotein (LDL) cholesterol levels in a subject to whom a describedAPOC3 RNAi agent has been administered is reduced by at least about 10%,20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, or greater than 99% relative to the subjectprior to being administered the APOC3 RNAi agent or to a subject notreceiving the APOC3 RNAi agent. The total cholesterol levels and/or LDLcholesterol levels in the subject may be reduced in a cell, group ofcells, tissue, blood, and/or other fluid of the subject.

A reduction in gene expression, mRNA, APOC3 protein levels, TG levels,cholesterol levels, and LDL cholesterol levels can be assessed by anymethods known in the art. As used herein, a reduction or decrease inAPOC3 mRNA level and/or protein level are collectively referred toherein as a reduction or decrease in APOC3 or inhibiting or reducing orknocking down the expression of APOC3. The Examples set forth hereinillustrate known methods for assessing inhibition of APOC3 geneexpression.

Cells, Tissues, Organs, and Non-Human Organisms

Cells, tissues, organs, and non-human organisms that include at leastone of the APOC3 RNAi agents described herein are contemplated. Thecell, tissue, organ, or non-human organism is made by delivering theRNAi agent to the cell, tissue, organ, or non-human organism.

The above provided embodiments and items are now illustrated with thefollowing, non-limiting examples.

EXAMPLES Example 1. Synthesis of APOC3 RNAi Agents

APOC3 RNAi agent duplexes shown in Table 3 and Table 6, above, weresynthesized in accordance with the following general procedures:

A. Synthesis.

The sense and antisense strands of the APOC3 RNAi agents weresynthesized according to phosphoramidite technology on solid phase usedin oligonucleotide synthesis. Depending on the scale, either aMerMade96E® (Bioautomation), a MerMadel2® (Bioautomation), or an OPPilot 100 (GE Healthcare) was used. Syntheses were performed on a solidsupport made of controlled pore glass (CPG, 500 Å or 600 Å, obtainedfrom Prime Synthesis, Aston, Pa., USA). All RNA and 2′-modified RNAphosphoramidites were purchased from Thermo Fisher Scientific(Milwaukee, Wis., USA). Specifically, the following 2′-O-methylphosphoramidites were used:(5′-O-dimethoxytrityl-N⁶-(benzoyl)-2′-O-methyl-adenosine-3′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite,5′-O-dimethoxy-trityl-N⁴-(acetyl)-2′-O-methyl-cytidine-3′-O-(2-cyanoethyl-N,N-diisopropyl-amino)phosphoramidite,(5′-O-dimethoxytrityl-N²-(isobutyryl)-2′-O-methyl-guanosine-3′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite, and5′-O-dimethoxytrityl-2′-O-methyl-uridine-3′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite. The 2′-deoxy-2′-fluoro-phosphoramidites carried thesame protecting groups as the 2′-O-methyl amidites.5′-dimethoxytrityl-2′-O-methyl-inosine-3′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidites were purchased from Glen Research (Virginia) or HongeneBiotech. The inverted abasic(3′-O-dimethoxytrityl-2′-deoxyribose-5′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidites were purchased from ChemGenes (Wilmington, Mass., USA).5′-(4,4′-Dimethoxytrityl)-2′,3′-seco-uridine,2′-benzoyl-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite wasalso purchased from Thermo Fisher Scientific or Hongene Biotech. The5′-O-dimethoxytrityl-N²,N⁶-(phenoxyacetate)-2′-O-methyl-diaminopurine-3′-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite was obtained from ChemGenes or Hongene Biotech.

Targeting ligand containing phosphoramidites were dissolved in anhydrousdichloromethane or anhydrous acetonitrile (50 mM), while all otheramidites were dissolved in anhydrous acetonitrile (50 mM), or anhydrousdimethylformamide and molecular sieves (3 Å) were added.5-Benzylthio-1H-tetrazole (BTT, 250 mM in acetonitrile) or5-Ethylthio-1H-tetrazole (ETT, 250 mM in acetonitrile) was used asactivator solution. Coupling times were 12 min (RNA), 15 min (targetingligand), 90 sec (2′OMe), and 60 sec (2′F). In order to introducephosphorothioate linkages, a_100 mM solution of 3-phenyl1,2,4-dithiazoline-5-one (POS, obtained from PolyOrg, Inc., Leominster,Mass., USA) in anhydrous Acetonitrile was employed. Unless specificallyidentified as a “naked” RNAi agent having no targeting ligand present,each of the APOC3 RNAi agent duplexes synthesized and tested in thefollowing Examples utilized N-acetyl-galactosamine as “NAG” in thetargeting ligand chemical structures represented in Table 7.

B. Cleavage and Deprotection of Support Bound Oligomer.

After finalization of the solid phase synthesis, the dried solid supportwas treated with a 1:1 volume solution of 40 wt. % methylamine in waterand 28% ammonium hydroxide solution (Aldrich) for 1.5 hours at 30° C.The solution was evaporated and the solid residue was reconstituted inwater (see below).

C. Purification.

Crude oligomers were purified by anionic exchange HPLC using a TSKgelSuperQ-5PW 13 μm column and Shimadzu LC-8 system. Buffer A was 20 mMTris, 5 mM EDTA, pH 9.0 and contained 20% Acetonitrile and buffer B wasthe same as buffer A with the addition of 1.5 M sodium chloride. UVtraces at 260 nm were recorded. Appropriate fractions were pooled thenrun on size exclusion HPLC using a GE Healthcare XK 26/40 column packedwith Sephadex G-25 fine with a running buffer of filtered DI water or100 mM ammonium bicarbonate, pH 6.7 and 20% Acetonitrile.

D. Annealing.

Complementary strands were mixed by combining equimolar RNA solutions(sense and antisense) in 1× Phosphate-Buffered Saline (Corning, Cellgro)to form the RNAi agents. Some RNAi agents were lyophilized and stored at−15 to −25° C. Duplex concentration was determined by measuring thesolution absorbance on a UV-Vis spectrometer in 1× Phosphate-BufferedSaline. The solution absorbance at 260 nm was then multiplied by aconversion factor and the dilution factor to determine the duplexconcentration. Unless otherwise stated, all conversion factor was 0.037mg/(mL·cm). For some experiments, a conversion factor was calculatedfrom an experimentally determined extinction coefficient.

Example 2. In Vitro Testing of APOC3 RNAi Agents

Candidate sequence duplexes shown in Table 3, above, were tested invitro. The APOC3 RNAi agents were prepared in accordance with theprocedures set forth in Example 1.

Evaluation of APOC3 RNAi agents in vitro was performed by transfectionof HuH7 cells, a human hepatocellular carcinoma line. Cells were platedat −7,500 cells per well in 96-well format, and each of the 65 APOC3RNAi agent duplexes shown in Table 3 was transfected at threeconcentrations (10 nM, 1 nM, and 0.1 nM), using LipoFectamine RNAiMax(Thermo Fisher) transfection reagent. Relative expression of each of theAPOC3 RNAi agents was determined by qRT-PCR by comparing the expressionlevels of APOC3 mRNA to an endogenous control, and normalized tountreated HuH7 cells (ΔΔC_(T) analysis), as shown in Table 8. Thus, forDuplex ID No. 56_1, average relative expression at 1 nM of 0.126 showsAPOC3 gene knockdown of 87.4%.

TABLE 8 In Vitro Testing of APOC3 RNAi Agents. Duplex Avg. Rel. High LowAvg. Rel. High Low Avg. Rel. High Low ID No. Exp. 10 nM (error) (error)Exp. 1 nM (error) (error) Exp. 0.1 nM (error) (error)  56_1 0.081 0.0130.016 0.126 0.033 0.045 0.249 0.083 0.125  56_2 0.076 0.014 0.017 0.1160.027 0.035 0.190 0.060 0.088  56_3 0.084 0.016 0.020 0.124 0.028 0.0360.313 0.114 0.179  56_4 0.098 0.023 0.031 0.155 0.045 0.063 0.534 0.1220.157  56_5 0.100 0.026 0.034 0.138 0.029 0.036 0.511 0.161 0.236  58_10.130 0.028 0.035 0.237 0.021 0.023 0.713 0.177 0.235  58_2 0.118 0.0180.021 0.319 0.039 0.045 0.602 0.058 0.064  58_3 0.070 0.011 0.013 0.1520.018 0.020 0.383 0.025 0.026  58_4 0.069 0.012 0.015 0.168 0.022 0.0250.453 0.031 0.034  58_5 0.062 0.009 0.011 0.189 0.047 0.062 0.557 0.0450.049 228_1 0.055 0.011 0.014 0.377 0.039 0.043 0.684 0.043 0.046 228_20.096 0.011 0.013 0.472 0.040 0.043 0.720 0.074 0.083 228_3 0.143 0.0230.027 0.525 0.021 0.022 0.804 0.035 0.036 228_4 0.115 0.018 0.022 0.5180.036 0.038 0.740 0.029 0.030 228_5 0.165 0.029 0.035 0.547 0.040 0.0430.721 0.036 0.038 235_1 0.142 0.025 0.030 0.566 0.045 0.049 0.737 0.0350.036 235_2 0.064 0.013 0.016 0.370 0.030 0.033 0.713 0.042 0.045 235_30.029 0.008 0.011 0.085 0.015 0.018 0.535 0.048 0.053 235_4 0.050 0.0100.012 0.197 0.018 0.019 0.652 0.045 0.048 235_5 0.079 0.019 0.025 0.3280.043 0.050 0.719 0.113 0.134 243_1 0.044 0.012 0.017 0.222 0.046 0.0580.671 0.114 0.137 243_2 0.035 0.008 0.011 0.358 0.041 0.047 0.701 0.0680.076 243_3 0.022 0.007 0.009 0.142 0.033 0.042 0.567 0.070 0.080 243_40.016 0.007 0.013 0.115 0.018 0.021 0.502 0.073 0.086 243_5 0.039 0.0110.016 0.123 0.019 0.022 0.597 0.050 0.055 260_1 0.021 0.007 0.011 0.3900.062 0.074 0.719 0.034 0.035 260_2 0.042 0.008 0.010 0.728 0.062 0.0680.719 0.042 0.045 260_3 0.026 0.008 0.012 0.747 0.067 0.073 0.685 0.0440.047 260_4 0.021 0.009 0.015 0.507 0.064 0.073 0.749 0.064 0.070 260_50.057 0.014 0.019 0.572 0.040 0.043 0.745 0.051 0.054 261_1 0.046 0.0070.008 0.295 0.039 0.045 0.766 0.044 0.046 261_2 0.052 0.017 0.024 0.6110.037 0.039 0.823 0.050 0.053 261_3 0.032 0.007 0.009 0.303 0.025 0.0280.727 0.024 0.025 261_4 0.027 0.005 0.007 0.756 0.031 0.032 0.690 0.0320.033 261_5 0.041 0.005 0.006 0.868 0.099 0.112 0.737 0.031 0.032 270_10.031 0.006 0.008 0.294 0.052 0.063 0.719 0.046 0.049 270_2 0.055 0.0150.020 0.344 0.066 0.082 0.738 0.036 0.038 270_3 0.047 0.014 0.019 0.3590.019 0.020 0.811 0.028 0.029 270_4 0.023 0.005 0.006 0.212 0.019 0.0210.706 0.034 0.035 270_5 0.027 0.007 0.010 0.615 0.030 0.032 0.685 0.0360.038 272_1 0.046 0.011 0.015 0.398 0.024 0.025 0.696 0.015 0.015 272_20.057 0.012 0.015 0.343 0.030 0.033 0.719 0.059 0.064 272_3 0.071 0.0100.012 0.269 0.034 0.039 0.736 0.034 0.036 272_4 0.061 0.018 0.026 0.1350.016 0.018 0.747 0.041 0.044 272_5 0.089 0.023 0.031 0.322 0.025 0.0270.793 0.029 0.030 273_1 0.014 0.004 0.006 0.066 0.019 0.026 0.665 0.0430.046 273_2 0.016 0.004 0.005 0.064 0.012 0.015 0.676 0.040 0.042 273_30.012 0.003 0.005 0.041 0.007 0.008 0.606 0.041 0.044 273_4 0.016 0.0030.004 0.060 0.009 0.011 0.687 0.036 0.038 273_5 0.024 0.004 0.004 0.1010.008 0.009 0.736 0.055 0.059 349_1 0.044 0.007 0.009 0.196 0.017 0.0180.711 0.091 0.104 349_2 0.054 0.017 0.025 0.226 0.015 0.016 0.820 0.0580.063 349_3 0.031 0.012 0.020 0.157 0.019 0.021 0.761 0.073 0.081 349_40.033 0.013 0.022 0.148 0.015 0.017 0.810 0.096 0.108 349_5 0.043 0.0160.024 0.214 0.013 0.014 0.853 0.077 0.084 434_1 0.074 0.008 0.009 0.1470.019 0.021 0.860 0.044 0.047 434_2 0.031 0.004 0.005 0.055 0.012 0.0160.390 0.080 0.101 434_3 0.026 0.003 0.004 0.053 0.011 0.015 0.418 0.0150.016 434_4 0.020 0.004 0.005 0.085 0.023 0.032 0.488 0.018 0.019 434_50.024 0.002 0.002 0.096 0.024 0.032 0.661 0.039 0.042 437_1 0.028 0.0050.006 0.073 0.022 0.031 0.689 0.033 0.034 437_2 0.046 0.006 0.006 0.1500.037 0.049 0.798 0.030 0.031 437_3 0.044 0.005 0.005 0.043 0.006 0.0070.591 0.023 0.024 437_4 0.023 0.004 0.006 0.030 0.005 0.006 0.759 0.0330.035 437_5 0.024 0.002 0.003 0.061 0.006 0.006 0.750 0.048 0.051

Example 3. APOC3-SEAP Mouse Model

Six to eight week old female C57BL/6 albino mice were transientlytransfected in vivo with plasmid by hydrodynamic tail vein injection,administered at least 15 days prior to administration of an APOC3 RNAiagent or control. The plasmid contains the APOC3 cDNA sequence (GenBankNM_000040.1 (SEQ ID NO: 1)) inserted into the 3′ UTR of the SEAP(secreted human placental alkaline phosphatase) reporter gene. 50 μg ofthe plasmid containing the APOC3 cDNA sequence in Ringer's Solution in atotal volume of 10% of the animal's body weight was injected into micevia the tail vein to create APOC3-SEAP model mice. The solution wasinjected through a 27-gauge needle in 5-7 seconds as previouslydescribed (Zhang G et al., “High levels of foreign gene expression inhepatocytes after tail vein injection of naked plasmid DNA.” Human GeneTherapy 1999 Vol. 10, p 1735-1737.). Inhibition of expression of APOC3by an APOC3 RNAi agent results in concomitant inhibition of SEAPexpression, which is measured. At day −1, SEAP expression levels inserum were measured by the Phospha-Light™ SEAP Reporter Gene AssaySystem (Invitrogen), and the mice were grouped according to average SEAPlevels.

Analyses:

SEAP levels may be measured at various times, both before and afteradministration of APOC3 RNAi agents.

-   -   i) Serum collection: Mice were anesthetized with 2-3% isoflurane        and blood samples were collected from the submandibular area        into serum separation tubes (Sarstedt AG & Co., Ntimbrecht,        Germany). Blood was allowed to coagulate at ambient temperature        for 20 min. The tubes were centrifuged at 8,000×g for 3 min to        separate the serum and stored at 4° C.    -   ii) Serum SEAP levels: Serum was collected and measured by the        Phospha-Light™ SEAP Reporter Gene Assay System (Invitrogen)        according to the manufacturer's instructions. Serum SEAP levels        for each animal was normalized to the control group of mice        injected with saline in order to account for the non-treatment        related decline in APOC3 expression with this model. First, the        SEAP level for each animal at a time point was divided by the        pre-treatment level of expression in that animal (Day −1) in        order to determine the ratio of expression “normalized to        pre-treatment”. Expression at a specific time point was then        normalized to the control group by dividing the “normalized to        pre-treatment” ratio for an individual animal by the average        “normalized to pre-treatment” ratio of all mice in the normal        saline control group. Alternatively, in some Examples set forth        herein, the serum SEAP levels for each animal were assessed by        normalizing to pre-treatment levels only.

Example 4. In Vivo Testing of APOC3 RNAi Agents in APOC3-SEAP Mice

The APOC3-SEAP mouse model described in Example 3, above, was used. Atday 1, each mouse was given a single subcutaneous administration of 200μl containing either 5 mg/kg (mpk) of an APOC3 RNAi agent, 3 mg/kg of anAPOC3 RNAi agent, or 200 μl of phosphate buffered saline without anAPOC3 RNAi agent to be used as a control, according to the followingTable 9.

TABLE 9 Dosing groups of APOC3-SEAP mice of Example 4. Group RNAi Agentand Dose Dosing Regimen A Saline (no RNAi agent) Single injection on day1 B 5.0 mg/kg AD04812 Single injection on day 1 C 5.0 mg/kg AD04813Single injection on day 1 D 5.0 mg/kg AD04814 Single injection on day 1E 3.0 mg/kg AD04814 Single injection on day 1 F 5.0 mg/kg AD04815 Singleinjection on day 1 G 5.0 mg/kg AD04816 Single injection on day 1 H 3.0mg/kg AD04816 Single injection on day 1 I 5.0 mg/kg AD04817 Singleinjection on day 1 J 5.0 mg/kg AD04818 Single injection on day 1 K 5.0mg/kg AD04819 Single injection on day 1 L 5.0 mg/kg AD04820 Singleinjection on day 1 M 5.0 mg/kg AD04821 Single injection on day 1

Each of the APOC3 RNAi agents included modified nucleotides that wereconjugated at the 5′ terminal end of the sense strand to a targetingligand that included three N-acetyl-galactosamine groups (tridentateligand) having the modified sequences as set forth in the duplexstructures herein. (See Tables 4, 5, 6, and 7 for specific modificationsand structure information related to the APOC3 RNAi agents).

The injections were performed between the skin and muscle (i.e.subcutaneous injections) into the loose skin over the neck and shoulderarea. Three (3) mice in each group were tested (n=3). Serum wascollected on day 8, day 15, day 22, and day 29, and SEAP expressionlevels were determined pursuant to the procedure set forth in Example 3,above. Data from the experiment is shown in the following Table 10, withAverage SEAP reflecting the normalized average value of SEAP:

TABLE 10 Average SEAP Normalized to Pre-Treatment and Saline Control inAPOC3-SEAP Mice from Example 4. Day 8 Day 15 Day 22 Day 29 Avg Std DevAvg Std Dev Avg Std Dev Avg Std Dev Group ID SEAP (+/−) SEAP (+/−) SEAP(+/−) SEAP (+/−) Group A (Saline) 1.000 0.157 1.000 0.603 1.000 0.8641.000 0.701 Group B (5.0 mg/kg AD04812) 0.112 0.009 0.112 0.009 0.0470.014 0.089 0.032 Group C (5.0 mg/kg AD04813) 0.091 0.009 0.046 0.0150.052 0.019 0.102 0.045 Group D (5.0 mg/kg AD04814) 0.065 0.023 0.0450.016 0.039 0.017 0.073 0.027 Group E (3.0 mg/kg AD04814) 0.075 0.0210.041 0.037 0.047 0.046 0.059 0.053 Group F (5.0 mg/kg AD04815) 0.0900.005 0.032 0.015 0.026 0.012 0.046 0.018 Group G (5.0 mg/kg AD04816)0.401 0.122 0.399 0.136 0.274 0.053 0.331 0.094 Group H (3.0 mg/kgAD04816) 0.389 0.129 0.292 0.090 0.218 0.070 0.185 0.039 Group I (5.0mg/kg AD04817) 0.371 0.210 0.266 0.091 0.098 0.014 0.144 0.033 Group J(5.0 mg/kg AD04818) 0.373 0.028 0.467 0.190 0.218 0.153 0.323 0.232Group K (5.0 mg/kg AD04819) 0.216 0.123 0.334 0.034 0.407 0.053 0.4080.042 Group L (5.0 mg/kg AD04820) 0.164 0.085 0.226 0.206 0.219 0.1650.252 0.157 Group M (5.0 mg/kg AD04821) 0.169 0.097 0.128 0.061 0.1500.105 0.191 0.143

Each of the APOC3 RNAi agents in each of the dosing groups (i.e., GroupsB through M) showed substantial reduction in SEAP as compared to thesaline control (Group A) across all measured time points. For example,APOC3 RNAi agent AD04815 exhibited approximately a 97.4% reduction inSEAP at day 22 after a single 5.0 mg/kg injection (0.026).

Example 5. APOC3 Transgenic Mouse Model

To assess and evaluate the effect of certain other APOC3 RNAi agents invivo, APOC3 transgenic mice were acquired commercially and used (TheJackson Laboratory, 006907—B6; CBA-Tg(APOC3)3707Bres/J). For APOC3transgenic mice, human APOC3 protein levels in serum were measured on aCobas® Integra 400 (Roche Diagnostics), according to the manufacturer'srecommendations.

For normalization, the APOC3 level for each animal at a time point wasdivided by the pre-treatment level of expression in that animal todetermine the ratio of expression “normalized to pre-dose”. In someExamples reported herein, the expression at a specific time point wasalso then normalized to the vehicle control group by dividing the“normalized to pre-dose” ratio for an individual animal by the mean“normalized to pre-dose” ratio of all mice in the vehicle control group.This resulted in expression for each time point normalized to that inthe control group.

APOC3 levels may be measured at various times, both before and afteradministration of APOC3 RNAi agents. Unless noted otherwise herein, micewere anesthetized with 2-3% isoflurane and blood samples were collectedfrom the submandibular area into serum separation tubes (Sarstedt AG &Co., Nimbrecht, Germany). Blood was allowed to coagulate at ambienttemperature for 20 min. The tubes were centrifuged at 8,000×g for 3 minto separate the serum and stored at 4° C.

Example 6. In Vivo Testing of APOC3 RNAi Agents in APOC3 Transgenic Mice

The APOC3 Transgenic Mouse Model described in Example 5, above, wasused. At day 1, each mouse was given a single subcutaneousadministration of 200 μl of the respective RNAi agent dissolved in D5W(dextrose in 5% water) or control (D5W), which included the dosinggroups shown in the following Table 11.

TABLE 11 Dosing Groups of APOC3 Transgenic Mice of Example 6. Group RNAiAgent and Dose Dosing Regimen A D5W (no RNAi agent) Single injection onday 1 B 4.0 mg/kg AD05172 Single injection on day 1 C 2.0 mg/kg AD05172Single injection on day 1 D 1.0 mg/kg AD05172 Single injection on day 1E 0.5 mg/kg AD05172 Single injection on day 1 F 1.0 mg/kg AD05215 Singleinjection on day 1 G 1.0 mg/kg AD05216 Single injection on day 1 H 1.0mg/kg AD05217 Single injection on day 1 I 1.0 mg/kg AD05218 Singleinjection on day 1 J 1.0 mg/kg AD05171 Single injection on day 1 K 2.0mg/kg AD05219 Single injection on day 1 L 2.0 mg/kg AD05222 Singleinjection on day 1 M 2.0 mg/kg AD05221 Single injection on day 1 N 2.0mg/kg AD05223 Single injection on day 1

Each of the APOC3 RNAi agents was conjugated to a targeting ligand thatincluded three N-acetyl-galactosamines (i.e., a tridentate NAG ligand),having the modified sequences and NAG structures as set forth herein.(See Tables 4, 5, 6, and 7 for specific modifications and structureinformation for the APOC3 RNAi agents used in Example 6).

The injections were performed between the skin and muscle (i.e.subcutaneous injections) into the loose skin over the neck and shoulderarea. Three (3) mice in each group were tested (n=3). Serum wascollected from the mice, including on day −1 (pre-dose bleed with a fourhour fast), and days 8, 15, 22, and 29. Mice were fasted for four hoursprior to each collection. APOC3 expression levels were determinedpursuant to the procedure set forth in Example 5, above. Data are shownin the following Table 12, with Average APOC3 reflecting the normalizedaverage value of APOC3 protein expressed in serum:

TABLE 12 Average APOC3 Protein Normalized to Pre-Treatment and VehicleControl (D5W) in APOC3 Transgenic Mice from Example 6. Day 8 Day 15 Day22 Day 29 Avg Std Dev Avg Std Dev Avg Std Dev Avg Std Dev Group ID APOC3(+/−) APOC3 (+/−) APOC3 (+/−) APOC3 (+/−) Group A (D5W) 1.000 0.0381.000 0.177 1.000 0.154 1.000 0.152 Group B (4.0 mg/kg AD05172) 0.0740.018 0.067 0.018 0.083 0.008 0.105 0.019 Group C (2.0 mg/kg AD05172)0.094 0.022 0.084 0.017 0.101 0.013 0.126 0.029 Group D (1.0 mg/kgAD05172) 0.113 0.039 0.115 0.038 0.150 0.050 0.212 0.095 Group E (0.5mg/kg AD05172) 0.153 0.050 0.191 0.087 0.245 0.102 0.461 0.169 Group F(1.0 mg/kg AD05215) 0.114 0.003 0.124 0.016 0.173 0.037 0.550 0.119Group G (1.0 mg/kg AD05216) 0.148 0.042 0.136 0.016 0.185 0.031 0.3420.034 Group H (1.0 mg/kg AD05217) 0.161 0.020 0.179 0.025 0.241 0.0480.464 0.306 Group I (1.0 mg/kg AD05218) 0.168 0.064 0.210 0.127 0.5170.248 0.779 0.418 Group J (1.0 mg/kg AD05171) 0.125 0.039 0.126 0.0430.165 0.050 0.302 0.117 Group K (2.0 mg/kg AD05219) 0.091 0.044 0.0700.018 0.084 0.025 0.095 0.034 Group L (2.0 mg/kg AD05222) 0.130 0.0540.230 0.114 0.265 0.147 0.484 0.047 Group M (2.0 mg/kg AD05221) 0.1310.026 0.148 0.041 0.289 0.126 0.410 0.098 Group N (2.0 mg/kg AD05223)0.082 0.047 0.062 0.019 0.073 0.021 0.080 0.022

Each of the APOC3 RNAi agents in each of the dosing groups (i.e., GroupsB through M) showed a reduction in APOC3 as compared to the control(Group A) across the measured time points. For example, after a single2.0 mg/kg dose on day 1, APOC3 RNAi agent AD05223 showed anapproximately 94% reduction (0.062) at day 15.

Example 7. In Vivo Testing of APOC3 RNAi Agents in APOC3 Transgenic Mice

The APOC3 Transgenic Mouse Model described in Example 5, above, wasused. At day 1, each mouse was given a single subcutaneousadministration of 200 μl of the respective RNAi agent dissolved in D5W(dextrose in 5% water) or control (D5W) according to the dosing groupsshown in the following Table 13.

TABLE 13 Dosing Groups of APOC3 Transgenic Mice of Example 7. Group RNAiAgent and Dose Dosing Regimen 1 D5W (no RNAi agent) Single injection onday 1 2 1.0 mg/kg AD05172 Single injection on day 1 3 1.0 mg/kg AD05255Single injection on day 1 4 1.0 mg/kg AD05169 Single injection on day 15 1.0 mg/kg AD05249 Single injection on day 1 6 1.0 mg/kg AD05250 Singleinjection on day 1 7 1.0 mg/kg AD05251 Single injection on day 1 8 1.0mg/kg AD05252 Single injection on day 1 9 1.0 mg/kg AD05253 Singleinjection on day 1 10 1.0 mg/kg AD05254 Single injection on day 1 11 1.0mg/kg AD05220 Single injection on day 1

Each of the APOC3 RNAi agents included modified nucleotides that wereconjugated at the 5′ terminal end of the sense strand to a targetingligand that included three N-acetyl-galactosamine groups (tridentateligand) having the modified sequences as set forth in the duplexstructures herein. (See Tables 4, 5, 6, and 7 for specific modificationsand structure information related to the APOC3 RNAi agents).

The injections were performed between the skin and muscle (i.e.subcutaneous injections) into the loose skin over the neck and shoulderarea. Three (3) mice in each group were tested (n=3). Serum wascollected from the mice, including on day −1 (pre-dose bleed with a fourhour fast), and days 8, 15, 22, and 29. Mice were fasted for four hoursprior to each collection. APOC3 expression levels were determinedpursuant to the procedure set forth in Example 5, above. Data from day 8of the experiment are shown in the following Table 14, with AverageAPOC3 reflecting the normalized average value of APOC3 protein expressedin serum:

TABLE 14 Average APOC3 Protein Normalized to Pre-Treatment and VehicleControl (D5W) in APOC3 Transgenic Mice from Example 7. Day 8 Day 15 Day22 Day 29 Avg Std Dev Avg Std Dev Avg Std Dev Avg Std Dev Group ID APOC3(+/−) APOC3 (+/−) APOC3 (+/−) APOC3 (+/−) Group 1 (D5W) 1.000 0.0921.000 0.096 1.000 0.089 1.000 0.103 Group 2 (1.0 mg/kg AD05172) 0.1250.033 0.133 0.040 0.175 0.050 0.198 0.061 Group 3 (1.0 mg/kg AD05255)N/A* N/A* 0.279 0.394 0.969 0.050 1.103 0.216 Group 4 (1.0 mg/kgAD05169) 0.179 0.056 0.185 0.067 0.206 0.058 0.245 0.084 Group 5 (1.0mg/kg AD05249) 0.212 0.045 0.263 0.055 0.460 0.083 0.863 0.586 Group 6(1.0 mg/kg AD05250) 0.167 0.070 0.146 0.048 0.169 0.062 0.203 0.051Group 7 (1.0 mg/kg AD05251) 0.140 0.071 0.121 0.077 0.170 0.094 0.1810.103 Group 8 (1.0 mg/kg AD05252) 0.143 0.045 0.167 0.050 0.184 0.0480.296 0.088 Group 9 (1.0 mg/kg AD05253) 0.192 0.068 0.202 0.063 0.2380.096 0.473 0.220 Group 10 (1.0 mg/kg AD05254) 0.184 0.075 0.225 0.0750.296 0.124 0.294 0.137 Group 11 (1.0 mg/kg AD05220) 0.089 0.012 0.1090.014 0.107 0.018 0.118 0.027 *samples for Group 3, Day 8 were lost dueto equipment failure

Each of the APOC3 RNAi agents in each of the dosing groups (i.e., Groups2 through 11) showed a reduction in APOC3 protein levels as compared tothe control (Group 1) at days 8 and 15. In particular, APOC3 RNAi agentsAD05251 and AD05169 (each having an antisense strand sequence designedto target position 438 of an APOC3 gene (i.e., SEQ ID NO:1), as well asAPOC3 RNA agent AD05220 (having an antisense strand sequence designed totarget position 506 of an APOC3 gene), showed particularly potentinhibitory effect. (See, e.g., Groups 4, 7, and 11 in Table 14, above).

Example 8. In Vivo Testing of APOC3 RNAi Agents in APOC3 Transgenic Mice

The APOC3 Transgenic Mouse Model described in Example 5, above, wasused. At day 1, each mouse was given a single subcutaneousadministration of 200 μl of the respective RNAi agent dissolved in D5W(dextrose in 5% water) or control (D5W) according to the dosing groupsshown in the following Table 13.

TABLE 15 Dosing groups of Example 8. Group RNAi Agent and Dose DosingRegimen 1 D5W (no RNAi agent) Single injection on day 1 2 0.5 mg/kgAD05540 Single injection on day 1 3 0.5 mg/kg AD05283 Single injectionon day 1 4 0.5 mg/kg AD05705 Single injection on day 1 5 0.5 mg/kgAD05706 Single injection on day 1 6 0.5 mg/kg AD05707 Single injectionon day 1 7 0.5 mg/kg AD05708 Single injection on day 1 8 0.5 mg/kgAD05709 Single injection on day 1 9 0.5 mg/kg AD05251 Single injectionon day 1 10 0.5 mg/kg AD05169 Single injection on day 1 11 0.5 mg/kgAD05710 Single injection on day 1 12 0.5 mg/kg AD05711 Single injectionon day 1 13 0.5 mg/kg AD05712 Single injection on day 1 14 0.5 mg/kgAD05713 Single injection on day 1 15 0.5 mg/kg AD05220 Single injectionon day 1 16 0.5 mg/kg AD05714 Single injection on day 1

Each of the APOC3 RNAi agents included modified nucleotides that wereconjugated at the 5′ terminal end of the sense strand to a targetingligand that included three N-acetyl-galactosamine groups (tridentateligand) having the modified sequences as set forth in the duplexstructures herein. (See Tables 4, 5, 6, and 7 for specific modificationsand structure information related to the APOC3 RNAi agents).

The injections were performed between the skin and muscle (i.e.subcutaneous injections) into the loose skin over the neck and shoulderarea. Three (3) mice in each Group were tested (n=3), except for Group 1(D5W vehicle) where four (4) mice were tested (n=4). Serum was collectedon day −1 (pre-dose bleed with a 4 hour fast), and days 8, and 15, 22,and 29. Mice were fasted for four hours prior to each collection. APOC3expression levels were determined pursuant to the procedure set forth inExample 5, above. Triglycerides, high-density lipoprotein (HDL),low-density lipoprotein (LDL), and total cholesterol in serum were alsomeasured on a Cobas® Integra 400 (Roche Diagnostics), according to themanufacturer's recommendations.

The APOC3 protein levels, triglyceride levels, HDL levels, and totalcholesterol levels for each animal were normalized. For normalization,the level of APOC3 protein, triglyceride, HDL, LDL, and totalcholesterol, respectively, for each animal at a time point, was dividedby the pre-treatment level of expression in that animal (in this case atday −1) to determine the ratio of expression “normalized topre-treatment.” Expression at a specific time point was then normalizedto the vehicle control group by dividing the “normalized topre-treatment” ratio for an individual animal by the mean “normalized topretreatment” ratio of all mice in the vehicle control group. Thisresulted in expression for each time point normalized to that in thecontrol group. Data from the experiment are shown in the followingTables 16 through 20:

TABLE 16 Average APOC3 Protein Normalized to Pre-Treatment and VehicleControl (D5W) from Example 8. Day 8 Day 15 Day 22 Day 29 Avg Std Dev AvgStd Dev Avg Std Dev Avg Std Dev Group ID APOC3 (+/−) APOC3 (+/−) APOC3(+/−) APOC3 (+/−) Group 1 (D5W) 1.000 0.104 1.000 0.297 1.000 0.3431.000 0.354 Group 2 (0.5 mg/kg AD05540) 0.196 0.020 0.203 0.044 0.2540.079 0.370 0.128 Group 3 (0.5 mg/kg AD05283) 0.178 0.077 0.195 0.0800.282 0.070 0.331 0.038 Group 4 (0.5 mg/kg AD05705) 0.146 0.053 0.1500.050 0.239 0.080 0.330 0.111 Group 5 (0.5 mg/kg AD05706) 0.153 0.0670.156 0.076 0.206 0.068 0.309 0.065 Group 6 (0.5 mg/kg AD05707) 0.1020.030 0.158 0.023 0.227 0.035 0.441 0.160 Group 7 (0.5 mg/kg AD05708)0.203 0.091 0.211 0.079 0.264 0.098 0.504 0.237 Group 8 (0.5 mg/kgAD05709) 0.213 0.086 0.190 0.078 0.299 0.143 0.467 0.250 Group 9 (0.5mg/kg AD05251) 0.170 0.062 0.142 0.062 0.138 0.073 0.184 0.061 Group 10(0.5 mg/kg AD05169) 0.290 0.131 0.320 0.054 0.309 0.039 0.433 0.060Group 11 (0.5 mg/kg AD05710) 0.379 0.024 0.481 0.146 0.696 0.116 0.7900.171 Group 12 (0.5 mg/kg AD05711) 0.331 0.028 0.325 0.036 0.334 0.0370.545 0.238 Group 13 (0.5 mg/kg AD05712) 0.208 0.058 0.223 0.130 0.2470.132 0.419 0.227 Group 14 (0.5 mg/kg AD05713) 0.216 0.092 0.305 0.1310.453 0.070 0.646 0.053 Group 15 (0.5 mg/kg AD05220) 0.232 0.104 0.1250.071 0.205 0.129 0.333 0.192 Group 16 (0.5 mg/kg AD05714) 0.338 0.0250.259 0.069 0.422 0.012 0.550 0.092

TABLE 17 Average Triglycerides Normalized to Pre-Treatment and VehicleControl (D5W) from Example 8. Day 8 Day 15 Day 22 Day 29 Avg Std Dev AvgStd Dev Avg Std Dev Avg Std Dev Group ID TG (+/−) TG (+/−) TG (+/−) TG(+/−) Group 1 (D5W) 1.000 0.279 1.000 0.454 1.000 0.423 1.000 0.440Group 2 (0.5 mg/kg AD05540) 0.232 0.041 0.218 0.072 0.264 0.111 0.3700.192 Group 3 (0.5 mg/kg AD05283) 0.222 0.154 0.225 0.153 0.319 0.1880.358 0.117 Group 4 (0.5 mg/kg AD05705) 0.141 0.036 0.123 0.033 0.2370.088 0.338 0.098 Group 5 (0.5 mg/kg AD05706) 0.154 0.073 0.145 0.0930.218 0.124 0.316 0.121 Group 6 (0.5 mg/kg AD05707) 0.109 0.049 0.1560.069 0.184 0.030 0.433 0.267 Group 7 (0.5 mg/kg AD05708) 0.279 0.1540.259 0.139 0.229 0.118 0.674 0.426 Group 8 (0.5 mg/kg AD05709) 0.2830.155 0.221 0.134 0.274 0.154 0.606 0.393 Group 9 (0.5 mg/kg AD05251)0.340 0.248 0.322 0.232 0.294 0.203 0.372 0.262 Group 10 (0.5 mg/kgAD05169) 0.274 0.202 0.306 0.078 0.276 0.062 0.341 0.118 Group 11 (0.5mg/kg AD05710) 0.360 0.087 0.409 0.197 0.700 0.155 0.707 0.276 Group 12(0.5 mg/kg AD05711) 0.268 0.096 0.288 0.061 0.293 0.054 0.488 0.248Group 13 (0.5 mg/kg AD05712) 0.170 0.068 0.171 0.100 0.213 0.127 0.4480.264 Group 14 (0.5 mg/kg AD05713) 0.183 0.088 0.262 0.148 0.399 0.0830.581 0.135 Group 15 (0.5 mg/kg AD05220) 0.208 0.121 0.081 0.048 0.2800.135 0.351 0.263 Group 16 (0.5 mg/kg AD05714) 0.319 0.082 0.242 0.1010.461 0.059 0.596 0.150

TABLE 18 Average Total Cholesterol Normalized to Pre-Treatment andVehicle Control (D5W) from Example 8. Day 8 Day 15 Day 22 Day 29 AvgTotal Std Dev Avg Total Std Dev Avg Total Std Dev Avg Total Std DevGroup ID Chol (+/−) Chol (+/−) Chol (+/−) Chol (+/−) Group 1 (D5W) 1.0000.063 1.000 0.370 1.000 0.386 1.000 0.335 Group 2 (0.5 mg/kg AD05540)0.414 0.103 0.464 0.144 0.483 0.179 0.583 0.214 Group 3 (0.5 mg/kgAD05283) 0.488 0.215 0.498 0.203 0.573 0.197 0.597 0.155 Group 4 (0.5mg/kg AD05705) 0.377 0.230 0.359 0.205 0.401 0.198 0.429 0.199 Group 5(0.5 mg/kg AD05706) 0.342 0.108 0.357 0.099 0.360 0.098 0.437 0.091Group 6 (0.5 mg/kg AD05707) 0.271 0.196 0.294 0.176 0.322 0.176 0.4410.235 Group 7 (0.5 mg/kg AD05708) 0.435 0.203 0.457 0.203 0.523 0.2300.629 0.290 Group 8 (0.5 mg/kg AD05709) 0.455 0.233 0.436 0.197 0.4540.216 0.590 0.321 Group 9 (0.5 mg/kg AD05251) 0.504 0.313 0.554 0.3450.533 0.327 0.636 0.398 Group 10 (0.5 mg/kg AD05169) 0.544 0.240 0.5950.285 0.538 0.235 0.578 0.155 Group 11 (0.5 mg/kg AD05710) 0.686 0.1380.810 0.240 0.916 0.185 0.987 0.242 Group 12 (0.5 mg/kg AD05711) 0.4930.105 0.457 0.094 0.483 0.076 0.658 0.222 Group 13 (0.5 mg/kg AD05712)0.414 0.214 0.440 0.258 0.416 0.227 0.556 0.322 Group 14 (0.5 mg/kgAD05713) 0.354 0.148 0.441 0.187 0.557 0.108 0.658 0.014 Group 15 (0.5mg/kg AD05220) 0.393 0.227 0.418 0.273 0.427 0.288 0.526 0.271 Group 16(0.5 mg/kg AD05714) 0.632 0.014 0.706 0.011 0.797 0.030 0.932 0.070

TABLE 19 Average HDL Normalized to Pre-Treatment and Vehicle Control(D5W) from Example 8. Day 8 Day 15 Day 22 Day 29 Avg Std Dev Avg Std DevAvg Std Dev Avg Std Dev Group ID HDL (+/−) HDL (+/−) HDL (+/−) HDL (+/−)Group 1 (D5W) 1.000 0.365 1.000 0.141 1.000 0.100 1.000 0.338 Group 2(0.5 mg/kg AD05540) 1.489 0.197 1.676 0.305 2.040 0.388 1.629 0.375Group 3 (0.5 mg/kg AD05283) 2.192 1.116 2.227 1.009 2.859 1.499 1.9820.785 Group 4 (0.5 mg/kg AD05705) 1.558 0.433 1.531 0.260 1.772 0.3340.953 0.316 Group 5 (0.5 mg/kg AD05706) 2.248 0.626 2.556 0.938 2.7360.875 1.878 0.629 Group 6 (0.5 mg/kg AD05707) 1.179 0.038 1.221 0.1621.352 0.204 1.100 0.266 Group 7 (0.5 mg/kg AD05708) 1.086 0.158 1.1870.252 1.670 0.203 0.972 0.400 Group 8 (0.5 mg/kg AD05709) 1.251 0.1871.308 0.280 1.519 0.299 1.000 0.346 Group 9 (0.5 mg/kg AD05251) 1.3370.326 1.369 0.372 1.961 0.901 1.426 0.438 Group 10 (0.5 mg/kg AD05169)1.239 0.023 1.050 0.436 1.180 0.633 1.242 0.416 Group 11 (0.5 mg/kgAD05710) 1.169 0.089 1.417 0.356 1.359 0.149 1.244 0.290 Group 12 (0.5mg/kg AD05711) 1.666 0.481 1.360 0.314 1.607 0.627 1.486 0.824 Group 13(0.5 mg/kg AD05712) 1.255 0.577 1.214 0.560 1.344 0.587 0.939 0.427Group 14 (0.5 mg/kg AD05713) 1.324 0.264 1.347 0.402 1.519 0.673 1.0470.507 Group 15 (0.5 mg/kg AD05220) 0.763 0.345 0.954 0.539 1.042 0.5330.963 0.093 Group 16 (0.5 mg/kg AD05714) 0.960 0.145 1.099 0.151 1.3820.108 1.124 0.022

TABLE 20 Average LDL Normalized to Pre-Treatment and Vehicle Control(D5W) from Example 8. Day 8 Day 15 Day 22 Day 29 Avg Std Dev Avg Std DevAvg Std Dev Avg Std Dev Group ID LDL (+/−) LDL (+/−) LDL (+/−) LDL (+/−)Group 1 (D5W) 1.000 0.314 1.000 0.350 1.000 0.448 1.000 0.268 Group 2(0.5 mg/kg AD05540) 0.265 0.076 0.318 0.100 0.340 0.104 0.517 0.199Group 3 (0.5 mg/kg AD05283) 0.404 0.201 0.426 0.209 0.560 0.292 0.5960.166 Group 4 (0.5 mg/kg AD05705) 0.303 0.245 0.271 0.209 0.315 0.1910.378 0.224 Group 5 (0.5 mg/kg AD05706) 0.226 0.101 0.272 0.056 0.2660.052 0.367 0.067 Group 6 (0.5 mg/kg AD05707) 0.160 0.128 0.204 0.1460.259 0.159 0.337 0.164 Group 7 (0.5 mg/kg AD05708) 0.251 0.130 0.2810.100 0.459 0.214 0.445 0.137 Group 8 (0.5 mg/kg AD05709) 0.242 0.1350.230 0.077 0.389 0.209 0.371 0.166 Group 9 (0.5 mg/kg AD05251) 0.4670.338 0.542 0.351 0.688 0.478 0.836 0.547 Group 10 (0.5 mg/kg AD05169)0.341 0.064 0.495 0.395 0.396 0.197 0.459 0.106 Group 11 (0.5 mg/kgAD05710) 0.742 0.257 0.997 0.398 0.944 0.357 1.228 0.474 Group 12 (0.5mg/kg AD05711) 0.526 0.135 0.401 0.116 0.737 0.388 0.919 0.367 Group 13(0.5 mg/kg AD05712) 0.373 0.156 0.423 0.182 0.440 0.193 0.477 0.294Group 14 (0.5 mg/kg AD05713) 0.312 0.159 0.370 0.144 0.736 0.194 1.0070.242 Group 15 (0.5 mg/kg AD05220) 0.369 0.164 0.337 0.204 0.401 0.2780.465 0.191 Group 16 (0.5 mg/kg AD05714) 0.440 0.062 0.500 0.055 0.7100.114 0.842 0.229

Each of the APOC3 RNAi agents in each of the dosing groups (i.e., Groups2 through 16) showed a reduction in APOC3 protein levels, triglyceridelevels, total cholesterol levels, and LDL levels as compared to thecontrol (Group 1). For example, a single 0.5 mg/kg dose of APOC3 RNAiagent AD05251 (Group 7) showed at day 22 a reduction of approximately86% of APOC3 protein levels (0.138), a reduction of approximately 70% intriglyceride levels (0.294), a reduction of approximately 47% of totalcholesterol levels (0.533), and a reduction of approximately 31% in LDLlevels (0.688). Further, as anticipated, on day 22 the administration ofAD05251 showed an increase in HDL levels (see, e.g., Table 19 above).

Example 9. In Vivo Dose Response Testing of APOC3 RNAi Agents in APOC3Transgenic Mice

The APOC3 Transgenic Mouse Model described in Example 5, above, wasused. At day 1, each mouse was given a single subcutaneousadministration of 200 μl of the respective RNAi agent dissolved in D5W(dextrose in 5% water) or control (D5W) according to the dosing groupsshown in the following Table 21:

TABLE 21 Dosing groups of Example 9. Group RNAi Agent and Dose DosingRegimen 1 D5W (no RNAi agent) Single injection on day 1 2 0.01 mg/kgAD05876 Single injection on day 1 3 0.05 mg/kg AD05876 Single injectionon day 1 4 0.1 mg/kg AD05876 Single injection on day 1 5 0.25 mg/kgAD05876 Single injection on day 1 6 0.5 mg/kg AD05876 Single injectionon day 1 7 1.0 mg/kg AD05876 Single injection on day 1 8 3.0 mg/kgAD05876 Single injection on day 1 9 0.01 mg/kg AD05251 Single injectionon day 1 10 0.05 mg/kg AD05251 Single injection on day 1 11 0.1 mg/kgAD05251 Single injection on day 1 12 0.25 mg/kg AD05251 Single injectionon day 1 13 0.5 mg/kg AD05251 Single injection on day 1 14 1.0 mg/kgAD05251 Single injection on day 1 15 3.0 mg/kg AD05251 Single injectionon day 1

Each of the APOC3 RNAi agents included modified nucleotides that wereconjugated at the 5′ terminal end of the sense strand to a targetingligand that included three N-acetyl-galactosamine groups (tridentateligand) having the structure of (NAG37)s. (See Tables 4, 5, 6, and 7 forspecific modifications and structure information related to the APOC3RNAi agents).

The injections were performed between the skin and muscle (i.e.subcutaneous injections) into the loose skin over the neck and shoulderarea. Four (4) mice in each Group were tested. Serum was collected onday −1 (pre-dose bleed with a 4 hour fast), and days 8, 15, 22, 29, and36. Mice were fasted for four hours prior to each collection. APOC3expression levels, triglycerides, high-density lipoprotein (HDL),low-density lipoprotein (LDL), and total cholesterol in serum weremeasured on a CobasR Integra 400 (Roche Diagnostics), according to themanufacturer's recommendations.

The APOC3 protein levels, triglyceride levels, HDL levels, and totalcholesterol levels for each animal were normalized. For normalization,the level of APOC3 protein, triglyceride, HDL, LDL, and totalcholesterol, respectively, for each animal at a time point, was dividedby the pre-treatment level of expression in that animal (in this case atday −1) to determine the ratio of expression “normalized to pre-dose.”Data from the experiment are shown in the following Tables 22 through26:

TABLE 22 Average APOC3 Protein Normalized to Pre-Dose from Example 9.Day 8 Day 15 Day 22 Day 29 Day 36 Avg Std Dev Avg Std Dev Avg Std DevAvg Std Dev Avg Std Dev Group ID APOC3 (+/−) APOC3 (+/−) APOC3 (+/−)APOC3 (+/−) APOC3 (+/−) Group 1 (D5W) 1.205 0.162 1.224 0.145 1.1020.257 1.011 0.148 1.103 0.133 Group 2 (0.01 mg/kg AD05876) 0.859 0.2550.970 0.231 1.050 0.101 1.001 0.091 0.990 0.121 Group 3 (0.05 mg/kgAD05876) 0.835 0.048 0.933 0.154 0.919 0.166 1.094 0.259 1.111 0.244Group 4 (0.1 mg/kg AD05876) 0.472 0.053 0.630 0.047 0.742 0.100 0.7980.117 0.937 0.064 Group 5 (0.25 mg/kg AD05876) 0.342 0.049 0.423 0.0450.495 0.056 0.734 0.066 0.812 0.097 Group 6 (0.5 mg/kg AD05876) 0.1880.030 0.211 0.045 0.289 0.029 0.386 0.047 0.504 0.050 Group 7 (1.0 mg/kgAD05876) 0.164 0.033 0.207 0.036 0.250 0.045 0.332 0.097 0.446 0.152Group 8 (3.0 mg/kg AD05876) 0.086 0.015 0.110 0.024 0.128 0.037 0.1410.023 0.157 0.031 Group 9 (0.01 mg/kg AD05251) 1.165 0.101 1.051 0.0400.955 0.105 1.038 0.033 0.968 0.079 Group 10 (0.05 mg/kg AD05251) 0.6750.051 0.694 0.056 0.692 0.046 0.836 0.139 0.921 0.087 Group 11 (0.1mg/kg AD05251) 0.590 0.098 0.478 0.073 0.562 0.067 0.625 0.054 0.6860.084 Group 12 (0.25 mg/kg AD05251) 0.273 0.067 0.295 0.039 0.354 0.0550.479 0.137 0.580 0.071 Group 13 (0.5 mg/kg AD05251) 0.219 0.066 0.2110.045 0.283 0.070 0.291 0.090 0.338 0.085 Group 14 (1.0 mg/kg AD05251)0.157 0.026 0.143 0.034 0.230 0.067 0.280 0.093 0.310 0.072 Group 15(3.0 mg/kg AD05251) 0.135 0.033 0.131 0.022 0.164 0.036 0.157 0.0480.191 0.056

TABLE 23 Average Triglycerides Normalized to Pre-Dose from Example 9.Day 8 Day 15 Day 22 Day 29 Day 36 Avg Std Dev Avg Std Dev Avg Std DevAvg Std Dev Avg Std Dev Group ID TG (+/−) TG (+/−) TG (+/−) TG (+/−) TG(+/−) Group 1 (D5W) 1.441 0.335 1.723 0.177 1.253 0.377 1.151 0.3011.304 0.221 Group 2 (0.01 mg/kg AD05876) 0.988 0.436 1.139 0.421 1.1770.271 1.209 0.242 1.259 0.325 Group 3 (0.05 mg/kg AD05876) 1.146 0.3031.321 0.459 0.964 0.355 1.428 0.613 1.275 0.456 Group 4 (0.1 mg/kgAD05876) 0.671 0.176 0.700 0.131 0.912 0.204 0.918 0.265 1.073 0.175Group 5 (0.25 mg/kg AD05876) 0.391 0.081 0.581 0.174 0.608 0.141 0.9600.205 0.989 0.196 Group 6 (0.5 mg/kg AD05876) 0.216 0.060 0.202 0.0540.306 0.092 0.465 0.147 0.493 0.066 Group 7 (1.0 mg/kg AD05876) 0.2270.099 0.326 0.147 0.366 0.096 0.427 0.150 0.600 0.261 Group 8 (3.0 mg/kgAD05876) 0.090 0.024 0.166 0.037 0.165 0.044 0.184 0.048 0.222 0.037Group 9 (0.01 mg/kg AD05251) 1.357 0.266 1.197 0.099 1.024 0.129 1.1970.101 1.118 0.215 Group 10 (0.05 mg/kg AD05251) 0.784 0.137 0.950 0.2780.725 0.137 1.013 0.270 1.108 0.257 Group 11 (0.1 mg/kg AD05251) 0.6340.182 0.583 0.110 0.587 0.160 0.641 0.123 0.702 0.172 Group 12 (0.25mg/kg AD05251) 0.330 0.119 0.397 0.076 0.393 0.042 0.583 0.236 0.6140.057 Group 13 (0.5 mg/kg AD05251) 0.250 0.084 0.197 0.040 0.283 0.0340.309 0.102 0.355 0.118 Group 14 (1.0 mg/kg AD05251) 0.213 0.054 0.1710.073 0.273 0.059 0.384 0.135 0.347 0.079 Group 15 (3.0 mg/kg AD05251)0.210 0.067 0.172 0.024 0.235 0.089 0.213 0.032 0.263 0.106

TABLE 24 Average Total Cholesterol Normalized to Pre-Dose from Example9. Day 8 Day 15 Day 22 Day 29 Day 36 Avg Total Std Dev Avg Total Std DevAvg Total Std Dev Avg Total Std Dev Avg Total Std Dev Group ID Chol(+/−) Chol (+/−) Chol (+/−) Chol (+/−) Chol (+/−) Group 1 (D5W) 1.1770.079 1.261 0.169 1.161 0.297 1.049 0.188 1.151 0.167 Group 2 (0.01mg/kg AD05876) 1.020 0.231 1.099 0.186 1.193 0.147 1.132 0.087 1.1410.157 Group 3 (0.05 mg/kg AD05876) 0.975 0.105 1.003 0.193 1.010 0.1921.169 0.296 1.160 0.265 Group 4 (0.1 mg/kg AD05876) 0.694 0.115 0.7490.101 0.851 0.122 0.876 0.155 1.005 0.063 Group 5 (0.25 mg/kg AD05876)0.670 0.188 0.744 0.229 0.792 0.190 0.953 0.116 0.928 0.157 Group 6 (0.5mg/kg AD05876) 0.556 0.146 0.600 0.178 0.628 0.127 0.672 0.126 0.7680.107 Group 7 (1.0 mg/kg AD05876) 0.596 0.081 0.634 0.145 0.664 0.1340.710 0.101 0.760 0.083 Group 8 (3.0 mg/kg AD05876) 0.547 0.057 0.5560.104 0.589 0.130 0.564 0.098 0.572 0.101 Group 9 (0.01 mg/kg AD05251)1.236 0.107 1.142 0.063 1.023 0.139 1.099 0.107 1.106 0.115 Group 10(0.05 mg/kg AD05251) 0.785 0.083 0.813 0.107 0.784 0.106 0.944 0.1470.995 0.135 Group 11 (0.1 mg/kg AD05251) 0.721 0.080 0.691 0.068 0.7060.065 0.737 0.028 0.814 0.060 Group 12 (0.25 mg/kg AD05251) 0.562 0.1150.617 0.104 0.632 0.081 0.705 0.076 0.777 0.044 Group 13 (0.5 mg/kgAD05251) 0.479 0.055 0.492 0.037 0.540 0.073 0.543 0.098 0.564 0.095Group 14 (1.0 mg/kg AD05251) 0.634 0.137 0.687 0.163 0.634 0.172 0.6690.163 0.700 0.174 Group 15 (3.0 mg/kg AD05251) 0.602 0.106 0.611 0.1010.632 0.121 0.627 0.167 0.594 0.121

TABLE 25 Average HDL Normalized to Pre-Dose from Example 9. Day 8 Day 15Day 22 Day 29 Day 36 Avg Std Dev Avg Std Dev Avg Std Dev Avg Std Dev AvgStd Dev Group ID HDL (+/−) HDL (+/−) HDL (+/−) HDL (+/−) HDL (+/−) Group1 (D5W) 0.883 0.115 0.855 0.013 0.919 0.081 1.070 0.128 0.905 0.109Group 2 (0.01 mg/kg AD05876) 1.029 0.087 1.086 0.209 0.987 0.191 1.0960.088 0.969 0.100 Group 3 (0.05 mg/kg AD05876) 0.786 0.184 0.968 0.1211.052 0.130 0.951 0.252 0.886 0.221 Group 4 (0.1 mg/kg AD05876) 1.1290.133 1.147 0.098 1.022 0.213 1.109 0.106 0.911 0.177 Group 5 (0.25mg/kg AD05876) 1.280 0.238 1.336 0.253 1.244 0.172 1.083 0.083 0.9920.082 Group 6 (0.5 mg/kg AD05876) 1.516 0.241 1.574 0.182 1.368 0.1851.327 0.172 1.350 0.237 Group 7 (1.0 mg/kg AD05876) 1.361 0.243 1.3270.318 1.298 0.173 1.330 0.208 1.206 0.262 Group 8 (3.0 mg/kg AD05876)1.620 0.459 1.452 0.347 1.542 0.371 1.477 0.227 1.417 0.322 Group 9(0.01 mg/kg AD05251) 0.833 0.143 0.808 0.133 0.856 0.154 0.936 0.1271.041 0.193 Group 10 (0.05 mg/kg AD05251) 1.036 0.111 0.913 0.017 1.0270.030 0.974 0.168 0.976 0.142 Group 11 (0.1 mg/kg AD05251) 1.075 0.0871.087 0.065 1.033 0.116 1.021 0.114 1.074 0.074 Group 12 (0.25 mg/kgAD05251) 1.118 0.094 1.175 0.062 1.100 0.051 1.142 0.146 1.152 0.113Group 13 (0.5 mg/kg AD05251) 1.344 0.178 1.455 0.124 1.329 0.190 1.3470.156 1.279 0.188 Group 14 (1.0 mg/kg AD05251) 1.338 0.143 1.501 0.1751.179 0.221 1.218 0.247 1.282 0.179 Group 15 (3.0 mg/kg AD05251) 1.3320.150 1.426 0.264 1.348 0.133 1.431 0.339 1.265 0.184

TABLE 26 Average LDL Normalized to Pre-Dose from Example 9. Day 8 Day 15Day 22 Day 29 Day 36 Avg Std Dev Avg Std Dev Avg Std Dev Avg Std Dev AvgStd Dev Group ID LDL (+/−) LDL (+/−) LDL (+/−) LDL (+/−) LDL (+/−) Group1 (D5W) 1.060 0.159 0.990 0.210 1.078 0.325 0.989 0.365 0.881 0.135Group 2 (0.01 mg/kg AD05876) 1.031 0.068 1.071 0.181 1.077 0.082 0.9920.154 0.958 0.081 Group 3 (0.05 mg/kg AD05876) 0.799 0.179 0.682 0.2230.859 0.177 0.959 0.289 0.954 0.176 Group 4 (0.1 mg/kg AD05876) 0.5350.019 0.593 0.071 0.636 0.145 0.692 0.100 0.840 0.089 Group 5 (0.25mg/kg AD05876) 0.645 0.153 0.570 0.152 0.660 0.158 0.783 0.083 0.6760.096 Group 6 (0.5 mg/kg AD05876) 0.624 0.238 0.645 0.192 0.620 0.0670.581 0.086 0.893 0.088 Group 7 (1.0 mg/kg AD05876) 0.481 0.124 0.4640.201 0.396 0.127 0.524 0.181 0.588 0.174 Group 8 (3.0 mg/kg AD05876)0.455 0.161 0.465 0.154 0.428 0.159 0.359 0.099 0.382 0.140 Group 9(0.01 mg/kg AD05251) 1.260 0.097 1.237 0.202 1.091 0.244 1.162 0.2091.356 0.249 Group 10 (0.05 mg/kg AD05251) 0.682 0.048 0.641 0.127 0.7150.032 0.792 0.123 0.847 0.223 Group 11 (0.1 mg/kg AD05251) 0.717 0.2930.635 0.146 0.693 0.260 0.679 0.234 0.845 0.128 Group 12 (0.25 mg/kgAD05251) 0.439 0.151 0.502 0.147 0.614 0.190 0.552 0.037 0.716 0.205Group 13 (0.5 mg/kg AD05251) 0.413 0.086 0.474 0.048 0.508 0.149 0.5420.209 0.514 0.162 Group 14 (1.0 mg/kg AD05251) 0.614 0.268 0.747 0.2920.601 0.266 0.633 0.282 0.669 0.271 Group 15 (3.0 mg/kg AD05251) 0.4880.162 0.469 0.099 0.498 0.176 0.445 0.230 0.405 0.142

Each of the APOC3 RNAi agents tested exhibited a dose response in thereduction of APOC3 protein levels, triglyceride levels, totalcholesterol levels, and LDL levels.

Example 10. In Vivo Dose Response Testing of APOC3 RNAi Agents in APOC3Transgenic Mice

The APOC3 Transgenic Mouse Model described in Example 5, above, wasused. At day 1, each mouse was given a single subcutaneousadministration of 200 μl of the respective RNAi agent dissolved in D5W(dextrose in 5% water) or control vehicle (D5W) according to the dosinggroups shown in the following Table 27:

TABLE 27 Dosing groups of Example 10. Group RNAi Agent and Dose DosingRegimen 1 D5W (no RNAi agent) Single injection on day 1 2 0.25 mg/kgAD05891 Single injection on day 1 3 0.25 mg/kg AD05892 Single injectionon day 1 4 0.25 mg/kg AD05893 Single injection on day 1 5 0.25 mg/kgAD05894 Single injection on day 1 6 0.25 mg/kg AD05895 Single injectionon day 1 7 0.25 mg/kg AD05896 Single injection on day 1 8 0.25 mg/kgAD05897 Single injection on day 1 9 0.25 mg/kg AD05889 Single injectionon day 1 10 0.25 mg/kg AD05890 Single injection on day 1 11 0.25 mg/kgAD05876 Single injection on day 1 12 0.25 mg/kg AD05877 Single injectionon day 1 13 0.25 mg/kg AD05878 Single injection on day 1 14 0.25 mg/kgAD05879 Single injection on day 1 15 0.25 mg/kg AD05880 Single injectionon day 1 16 0.25 mg/kg AD05882 Single injection on day 1 17 0.25 mg/kgAD05884 Single injection on day 1 18 0.25 mg/kg AD05885 Single injectionon day 1 19 0.25 mg/kg AD05886 Single injection on day 1 20 0.25 mg/kgAD05887 Single injection on day 1 21 0.25 mg/kg AD05888 Single injectionon day 1 22 0.25 mg/kg AD05769 Single injection on day 1

Each of the APOC3 RNAi agents included modified nucleotides that wereconjugated at the 5′ terminal end of the sense strand to a targetingligand that included three N-acetyl-galactosamine groups (tridentateligand) having the modified sequences as set forth in the duplexstructures herein. (See Tables 4, 5, 6, and 7 for specific modificationsand structure information related to the APOC3 RNAi agents).

The APOC3 RNAi agents tested in Example 10 included nucleotide sequencesthat were designed to target different positions on the APOC3 gene(i.e., SEQ ID NO:1). More specifically, Groups 2-4 (i.e., APOC3 RNAiagents AD05891, AD05892, and AD05893) included antisense strandsequences designed to target position 248 of an APOC3 gene; Group 5(i.e., APOC3 RNAi agent AD05894) included an antisense strand sequencedesigned to target position 263 of an APOC3 gene; Groups 6-7 (i.e.,APOC3 RNAi agents AD05895 and AD05896) included antisense strandsequences designed to target position 422 of an APOC3 gene; Group 8(i.e., APOC3 RNAi agent AD05897) included an antisense strand sequencedesigned to target position 246 of an APOC3 gene; Groups 9-10 (i.e.,APOC3 RNAi agents AD05889 and AD05890) included antisense strandsequences designed to target position 168 of an APOC3 gene; and Groups11-22 (i.e., APOC3 RNAi agents AD05876, AD05877, AD05878, AD05878,AD05880, AD05882, AD05884, AD05885, AD05886, AD05887, AD05888, andAD05769) included antisense strand sequences designed to target position438 of an APOC3 gene.

The injections were performed between the skin and muscle (i.e.subcutaneous injections) into the loose skin over the neck and shoulderarea. Three (3) mice in each Group were tested (n=3). Serum wascollected on day −1 (pre-dose bleed with a 4 hour fast), and days 8, 15.For mice dosed with the certain RNAi agents that exhibited relativelyhigh inhibitory activity and for the mice dosed with the vehiclecontrol, additional serum samples were collected on days 22 and 29. Micewere fasted for four hours prior to each collection. APOC3 expressionlevels, triglycerides, high-density lipoprotein (HDL), low-densitylipoprotein (LDL), and total cholesterol in serum were measured on aCobasR Integra 400 (Roche Diagnostics), according to the manufacturer'srecommendations.

The APOC3 protein levels, triglyceride levels, HDL levels, and totalcholesterol levels for each animal were normalized. For normalization,the level of APOC3 protein, triglyceride, HDL, LDL, and totalcholesterol, respectively, for each animal at a time point, was dividedby the pre-treatment level of expression in that animal (in this case atday −1) to determine the ratio of expression “normalized to pre-dose.”Data from the experiment are shown in the following Tables 28 through32:

TABLE 28 Average APOC3 Protein Normalized to Pre-Dose from Example 10.Day 8 Day 15 Day 22 Day 29 Avg Std Dev Avg Std Dev Avg Std Dev Avg StdDev Group ID APOC3 (+/−) APOC3 (+/−) APOC3 (+/−) APOC3 (+/−) Group 1(D5W) 1.130 0.131 0.892 0.155 1.182 0.272 1.126 0.174 Group 2 (0.25mg/kg AD05891) 0.944 0.060 0.874 0.037 N/A N/A N/A N/A Group 3 (0.25mg/kg AD05892) 0.831 0.101 0.840 0.116 N/A N/A N/A N/A Group 4 (0.25mg/kg AD05893) 1.030 0.030 1.020 0.137 N/A N/A N/A N/A Group 5 (0.25mg/kg AD05894) 0.835 0.136 0.774 0.134 N/A N/A N/A N/A Group 6 (0.25mg/kg AD05895) 0.771 0.186 0.632 0.157 N/A N/A N/A N/A Group 7 (0.25mg/kg AD05896) 0.912 0.109 0.836 0.218 N/A N/A N/A N/A Group 8 (0.25mg/kg AD05897) 0.726 0.102 0.777 0.134 N/A N/A N/A N/A Group 9 (0.25mg/kg AD05889) 1.059 0.187 0.987 0.123 N/A N/A N/A N/A Group 10 (0.25mg/kg AD05890) 0.984 0.091 1.119 0.145 N/A N/A N/A N/A Group 11 (0.25mg/kg AD05876) 0.222 0.021 0.258 0.034 0.361 0.027 0.523 0.126 Group 12(0.25 mg/kg AD05877) 0.457 0.034 0.392 0.065 0.492 0.134 N/A N/A Group13 (0.25 mg/kg AD05878) 0.366 0.115 0.406 0.134 0.567 0.232 N/A N/AGroup 14 (0.25 mg/kg AD05879) 0.560 0.082 0.493 0.121 0.679 0.085 N/AN/A Group 15 (0.25 mg/kg AD05880) 0.572 0.205 0.652 0.274 N/A N/A N/AN/A Group 16 (0.25 mg/kg AD05882) 1.117 0.230 1.160 0.188 N/A N/A N/AN/A Group 17 (0.25 mg/kg AD05884) 0.425 0.103 0.444 0.158 0.580 0.180N/A N/A Group 18 (0.25 mg/kg AD05885) 0.629 0.024 0.782 0.109 N/A N/AN/A N/A Group 19 (0.25 mg/kg AD05886) 1.041 0.474 1.256 0.634 N/A N/AN/A N/A Group 20 (0.25 mg/kg AD05887) 0.390 0.106 0.608 0.159 N/A N/AN/A N/A Group 21 (0.25 mg/kg AD05888) 0.429 0.107 0.591 0.105 N/A N/AN/A N/A Group 22 (0.25 mg/kg AD05769) 0.229 0.039 0.346 0.078 0.3250.061 0.407 0.017

TABLE 29 Average Triglycerides Normalized to Pre-Dose from Example 10.Day 8 Day 15 Day 22 Day 29 Avg Std Dev Avg Std Dev Avg Std Dev Avg StdDev Group ID TG (+/−) TG (+/−) TG (+/−) TG (+/−) Group 1 (D5W) 1.3570.213 0.942 0.220 1.389 0.468 1.225 0.268 Group 2 (0.25 mg/kg AD05891)1.123 0.127 0.908 0.057 N/A N/A N/A N/A Group 3 (0.25 mg/kg AD05892)0.924 0.039 0.859 0.202 N/A N/A N/A N/A Group 4 (0.25 mg/kg AD05893)1.262 0.056 1.168 0.189 N/A N/A N/A N/A Group 5 (0.25 mg/kg AD05894)0.903 0.297 0.834 0.239 N/A N/A N/A N/A Group 6 (0.25 mg/kg AD05895)0.728 0.300 0.632 0.207 N/A N/A N/A N/A Group 7 (0.25 mg/kg AD05896)0.929 0.107 0.907 0.268 N/A N/A N/A N/A Group 8 (0.25 mg/kg AD05897)0.836 0.178 0.936 0.212 N/A N/A N/A N/A Group 9 (0.25 mg/kg AD05889)1.162 0.270 1.096 0.270 N/A N/A N/A N/A Group 10 (0.25 mg/kg AD05890)0.992 0.341 1.486 0.505 N/A N/A N/A N/A Group 11 (0.25 mg/kg AD05876)0.234 0.054 0.316 0.091 0.333 0.026 0.581 0.203 Group 12 (0.25 mg/kgAD05877) 0.496 0.096 0.530 0.175 0.653 0.215 N/A N/A Group 13 (0.25mg/kg AD05878) 0.450 0.214 0.619 0.314 0.781 0.434 N/A N/A Group 14(0.25 mg/kg AD05879) 0.664 0.033 0.664 0.072 0.905 0.030 N/A N/A Group15 (0.25 mg/kg AD05880) 0.726 0.384 0.790 0.399 N/A N/A N/A N/A Group 16(0.25 mg/kg AD05882) 1.289 0.436 1.695 0.408 N/A N/A N/A N/A Group 17(0.25 mg/kg AD05884) 0.376 0.132 0.554 0.283 0.605 0.296 N/A N/A Group18 (0.25 mg/kg AD05885) 0.620 0.064 0.998 0.219 N/A N/A N/A N/A Group 19(0.25 mg/kg AD05886) 1.315 0.665 1.941 1.267 N/A N/A N/A N/A Group 20(0.25 mg/kg AD05887) 0.445 0.193 0.867 0.335 N/A N/A N/A N/A Group 21(0.25 mg/kg AD05888) 0.467 0.227 0.700 0.190 N/A N/A N/A N/A Group 22(0.25 mg/kg AD05769) 0.204 0.033 0.377 0.068 0.373 0.097 0.370 0.071

TABLE 30 Average Total Cholesterol Normalized to Pre-Dose from Example10. Day 8 Day 15 Day 22 Day 29 Avg Total Std Dev Avg Total Std Dev AvgTotal Std Dev Avg Total Std Dev Group ID Chol (+/−) Chol (+/−) Chol(+/−) Chol (+/−) Group 1 (D5W) 1.186 0.199 0.761 0.107 1.131 0.325 1.2030.267 Group 2 (0.25 mg/kg AD05891) 1.056 0.104 0.947 0.161 N/A N/A N/AN/A Group 3 (0.25 mg/kg AD05892) 0.860 0.111 0.856 0.142 N/A N/A N/A N/AGroup 4 (0.25 mg/kg AD05893) 1.132 0.037 1.137 0.163 N/A N/A N/A N/AGroup 5 (0.25 mg/kg AD05894) 0.776 0.145 0.795 0.144 N/A N/A N/A N/AGroup 6 (0.25 mg/kg AD05895) 0.852 0.275 0.808 0.220 N/A N/A N/A N/AGroup 7 (0.25 mg/kg AD05896) 0.995 0.080 0.943 0.114 N/A N/A N/A N/AGroup 8 (0.25 mg/kg AD05897) 0.978 0.160 1.015 0.136 N/A N/A N/A N/AGroup 9 (0.25 mg/kg AD05889) 1.094 0.205 1.018 0.166 N/A N/A N/A N/AGroup 10 (0.25 mg/kg AD05890) 1.032 0.055 1.015 0.196 N/A N/A N/A N/AGroup 11 (0.25 mg/kg AD05876) 0.573 0.180 0.565 0.117 0.657 0.107 0.7820.052 Group 12 (0.25 mg/kg AD05877) 0.673 0.141 0.595 0.156 0.688 0.235N/A N/A Group 13 (0.25 mg/kg AD05878) 0.598 0.231 0.609 0.227 0.6890.293 N/A N/A Group 14 (0.25 mg/kg AD05879) 0.705 0.052 0.655 0.0410.848 0.111 N/A N/A Group 15 (0.25 mg/kg AD05880) 0.596 0.230 0.6350.235 N/A N/A N/A N/A Group 16 (0.25 mg/kg AD05882) 1.169 0.241 1.2680.327 N/A N/A N/A N/A Group 17 (0.25 mg/kg AD05884) 0.597 0.290 0.5740.254 0.668 0.284 N/A N/A Group 18 (0.25 mg/kg AD05885) 0.765 0.1920.837 0.089 N/A N/A N/A N/A Group 19 (0.25 mg/kg AD05886) 1.043 0.2851.336 0.497 N/A N/A N/A N/A Group 20 (0.25 mg/kg AD05887) 0.679 0.0870.843 0.174 N/A N/A N/A N/A Group 21 (0.25 mg/kg AD05888) 0.674 0.2920.807 0.302 N/A N/A N/A N/A Group 22 (0.25 mg/kg AD05769) 0.479 0.0940.551 0.122 0.537 0.075 0.583 0.125

TABLE 31 Average HDL Normalized to Pre-Dose from Example 10. Day 8 Day15 Day 22 Day 29 Avg Std Dev Avg Std Dev Avg Std Dev Avg Std Dev GroupID HDL (+/−) HDL (+/−) HDL (+/−) HDL (+/−) Group 1 (D5W) 0.837 0.0620.761 0.079 0.697 0.019 0.910 0.079 Group 2 (0.25 mg/kg AD05891) 0.6680.206 0.809 0.267 N/A N/A N/A N/A Group 3 (0.25 mg/kg AD05892) 0.6120.231 0.833 0.182 N/A N/A N/A N/A Group 4 (0.25 mg/kg AD05893) 0.7790.343 0.820 0.331 N/A N/A N/A N/A Group 5 (0.25 mg/kg AD05894) 0.8560.148 0.942 0.212 N/A N/A N/A N/A Group 6 (0.25 mg/kg AD05895) 1.2350.117 1.241 0.079 N/A N/A N/A N/A Group 7 (0.25 mg/kg AD05896) 1.2790.792 1.248 0.740 N/A N/A N/A N/A Group 8 (0.25 mg/kg AD05897) 1.1220.285 0.992 0.298 N/A N/A N/A N/A Group 9 (0.25 mg/kg AD05889) 0.7830.278 0.718 0.203 N/A N/A N/A N/A Group 10 (0.25 mg/kg AD05890) 0.8850.294 0.661 0.131 N/A N/A N/A N/A Group 11 (0.25 mg/kg AD05876) 2.0590.818 1.747 0.597 1.981 0.319 1.748 0.825 Group 12 (0.25 mg/kg AD05877)1.317 0.148 1.295 0.273 1.176 0.130 N/A N/A Group 13 (0.25 mg/kgAD05878) 1.421 0.294 1.273 0.262 0.999 0.328 N/A N/A Group 14 (0.25mg/kg AD05879) 1.037 0.074 0.945 0.125 0.924 0.141 N/A N/A Group 15(0.25 mg/kg AD05880) 0.905 0.266 0.855 0.051 N/A N/A N/A N/A Group 16(0.25 mg/kg AD05882) 0.784 0.098 0.621 0.103 N/A N/A N/A N/A Group 17(0.25 mg/kg AD05884) 1.529 0.486 1.228 0.309 1.149 0.257 N/A N/A Group18 (0.25 mg/kg AD05885) 1.123 0.323 0.651 0.143 N/A N/A N/A N/A Group 19(0.25 mg/kg AD05886) 1.047 0.343 0.675 0.181 N/A N/A N/A N/A Group 20(0.25 mg/kg AD05887) 2.093 1.089 1.487 0.748 N/A N/A N/A N/A Group 21(0.25 mg/kg AD05888) 1.452 0.065 1.245 0.177 N/A N/A N/A N/A Group 22(0.25 mg/kg AD05769) 1.289 0.219 1.186 0.202 1.125 0.231 1.325 0.044

TABLE 32 Average LDL Normalized to Pre-Dose from Example 10. Day 8 Day15 Day 22 Day 29 Avg Std Dev Avg Std Dev Avg Std Dev Avg Std Dev GroupID LDL (+/−) LDL (+/−) LDL (+/−) LDL (+/−) Group 1 (D5W) 1.456 0.3911.107 0.243 1.506 0.751 1.568 0.650 Group 2 (0.25 mg/kg AD05891) 1.4170.351 1.593 0.488 N/A N/A N/A N/A Group 3 (0.25 mg/kg AD05892) 0.8750.398 0.951 0.170 N/A N/A N/A N/A Group 4 (0.25 mg/kg AD05893) 1.2430.217 1.400 0.311 N/A N/A N/A N/A Group 5 (0.25 mg/kg AD05894) 0.7760.175 0.934 0.244 N/A N/A N/A N/A Group 6 (0.25 mg/kg AD05895) 1.2230.413 1.208 0.361 N/A N/A N/A N/A Group 7 (0.25 mg/kg AD05896) 1.3470.475 1.224 0.331 N/A N/A N/A N/A Group 8 (0.25 mg/kg AD05897) 1.2060.398 1.255 0.137 N/A N/A N/A N/A Group 9 (0.25 mg/kg AD05889) 1.2910.294 1.329 0.267 N/A N/A N/A N/A Group 10 (0.25 mg/kg AD05890) 1.1710.363 1.091 0.284 N/A N/A N/A N/A Group 11 (0.25 mg/kg AD05876) 0.6790.457 0.703 0.329 0.881 0.237 0.896 0.252 Group 12 (0.25 mg/kg AD05877)0.575 0.162 0.531 0.187 0.624 0.304 N/A N/A Group 13 (0.25 mg/kgAD05878) 0.534 0.191 0.532 0.163 0.666 0.321 N/A N/A Group 14 (0.25mg/kg AD05879) 0.602 0.043 0.671 0.060 0.939 0.171 N/A N/A Group 15(0.25 mg/kg AD05880) 0.527 0.098 0.525 0.122 N/A N/A N/A N/A Group 16(0.25 mg/kg AD05882) 1.252 0.279 1.568 0.525 N/A N/A N/A N/A Group 17(0.25 mg/kg AD05884) 0.814 0.591 0.590 0.363 0.850 0.453 N/A N/A Group18 (0.25 mg/kg AD05885) 0.827 0.171 0.798 0.043 N/A N/A N/A N/A Group 19(0.25 mg/kg AD05886) 1.045 0.206 1.180 0.134 N/A N/A N/A N/A Group 20(0.25 mg/kg AD05887) 0.756 0.118 0.794 0.156 N/A N/A N/A N/A Group 21(0.25 mg/kg AD05888) 0.745 0.460 0.945 0.499 N/A N/A N/A N/A Group 22(0.25 mg/kg AD05769) 0.634 0.293 0.568 0.243 0.625 0.189 0.644 0.136

As shown in Tables 28-32 above, the RNAi agents in Groups 2 through 10(i.e., RNAi agents with antisense strands designed to target an APOC3gene at positions 248, 263, 422, 246, and 168) showed relatively limitedinhibitory effect, particularly when compared to the RNAi agents inGroups 11 through 22, which all included antisense strand nucleotidesequences designed to target position 438 of an APOC3 gene. Further, ofthose RNAi agents that included sequences targeting position 438 of theAPOC3 gene, Group 11 (AD05876) and Group 22 (AD05769) showed thegreatest level of inhibitory effect with respect to APOC3 proteinlevels, triglycerides, and total cholesterol levels.

Example 11. In Vivo Testing of APOC3 RNAi Agents in APOC3 TransgenicMice

The APOC3 Transgenic Mouse Model described in Example 5, above, wasused. At day 1, each mouse was given a single subcutaneousadministration of 200 μl of the respective RNAi agent dissolved in D5W(dextrose in 5% water) or control vehicle (D5W) according to the dosinggroups shown in the following Table 33:

TABLE 33 Dosing groups of Example 11. Group RNAi Agent and Dose DosingRegimen 1 D5W (no RNAi agent) Single injection on day 1 2 0.5 mg/kgAD05260 Single injection on day 1 3 0.5 mg/kg AD05221 Single injectionon day 1 4 0.5 mg/kg AD05223 Single injection on day 1 5 0.5 mg/kgAD05299 Single injection on day 1 6 0.5 mg/kg AD05283 Single injectionon day 1 7 0.5 mg/kg AD05284 Single injection on day 1 8 0.5 mg/kgAD05167 Single injection on day 1 9 0.5 mg/kg AD05168 Single injectionon day 1 10 0.5 mg/kg AD05171 Single injection on day 1 11 0.5 mg/kgAD05258 Single injection on day 1 12 0.5 mg/kg AD05259 Single injectionon day 1 13 0.5 mg/kg AD05169 Single injection on day 1 14 0.5 mg/kgAD05239 Single injection on day 1 15 0.5 mg/kg AD05251 Single injectionon day 1 16 0.5 mg/kg AD05220 Single injection on day 1

Each of the APOC3 RNAi agents included modified nucleotides that wereconjugated at the 5′ terminal end of the sense strand to a targetingligand that included three N-acetyl-galactosamine groups (tridentateligand) having the modified sequences as set forth in the duplexstructures herein. (See Tables 4, 5, 6, and 7 for specific modificationsand structure information related to the APOC3 RNAi agents).

The APOC3 RNAi agents tested in Example 11 included nucleotide sequencesthat were designed to target different positions on the APOC3 gene(i.e., SEQ ID NO:1). More specifically, Group 2 (i.e., APOC3 RNAi agentAD05260) included an antisense strand sequence designed to targetposition 58 of an APOC3 gene; Group 3 (i.e., APOC3 RNAi agent AD05221)included an antisense strand sequence designed to target position 246 ofan APOC3 gene; Groups 4-7 (i.e., APOC3 RNAi agents AD05223, AD05299,AD05283, and AD05284) included antisense strand sequences designed totarget position 432 of an APOC3 gene; Groups 8-12 (i.e., APOC3 RNAiagents AD05167, AD05168, AD05171, AD05258, and AD05259) includedantisense strand sequences designed to target position 434 of an APOC3gene; Groups 13-15 (i.e., APOC3 RNAi agents AD05169, AD05239, andAD05251) included antisense strand sequences designed to target position438 of an APOC3 gene; and Group 16 (i.e., APOC3 RNAi agent AD05220)included an antisense strand sequence designed to target position 506 ofan APOC3 gene.

The injections were performed between the skin and muscle (i.e.subcutaneous injections) into the loose skin over the neck and shoulderarea. Three (3) mice in each Group were tested (n=3). Serum wascollected on day −1 (pre-dose bleed with a 4 hour fast), and days 8, 15.For mice dosed with the certain RNAi agents that exhibited relativelyhigh inhibitory activity and for the mice dosed with the vehiclecontrol, additional serum samples were collected on days 22 and 29. Micewere fasted for four hours prior to each collection. APOC3 expressionlevels, triglycerides, high-density lipoprotein (HDL), low-densitylipoprotein (LDL), and total cholesterol in serum were measured on aCobasR Integra 400 (Roche Diagnostics), according to the manufacturer'srecommendations.

The APOC3 protein levels, triglyceride levels, HDL levels, and totalcholesterol levels for each animal were normalized. For normalization,the level of APOC3 protein, triglyceride, HDL, LDL, and totalcholesterol, respectively, for each animal at a time point, was dividedby the pre-treatment level of expression in that animal (in this case atday −1) to determine the ratio of expression “normalized to pre-dose.”Data from the experiment are shown in the following Tables 34 through38:

TABLE 34 Average APOC3 Protein Normalized to Pre-Dose from Example 11.Day 8 Day 15 Day 22 Day 29 Avg Std Dev Avg Std Dev Avg Std Dev Avg StdDev Group ID APOC3 (+/−) APOC3 (+/−) APOC3 (+/−) APOC3 (+/−) Group 1(D5W) 0.854 0.102 0.866 0.140 0.881 0.079 0.857 0.140 Group 2 (0.5 mg/kgAD05260) 0.297 0.031 0.352 0.042 N/A N/A N/A N/A Group 3 (0.5 mg/kgAD05221) 0.483 0.060 0.619 0.046 N/A N/A N/A N/A Group 4 (0.5 mg/kgAD05223) 0.123 0.048 0.242 0.101 0.311 0.099 0.424 0.152 Group 5 (0.5mg/kg AD05299) 0.272 0.047 0.589 0.016 N/A N/A N/A N/A Group 6 (0.5mg/kg AD05283) 0.108 0.014 0.121 0.011 0.163 0.009 0.201 0.032 Group 7(0.5 mg/kg AD05284) 0.174 0.111 0.208 0.123 0.313 0.124 0.405 0.144Group 8 (0.5 mg/kg AD05167) 0.466 0.093 0.656 0.286 N/A N/A N/A N/AGroup 9 (0.5 mg/kg AD05168) 0.146 0.046 0.452 0.098 N/A N/A N/A N/AGroup 10 (0.5 mg/kg AD05171) 0.191 0.088 0.199 0.095 0.419 0.070 0.5480.087 Group 11 (0.5 mg/kg AD05258) 0.545 0.147 0.624 0.142 N/A N/A N/AN/A Group 12 (0.5 mg/kg AD05259) 0.236 0.047 0.300 0.115 N/A N/A N/A N/AGroup 13 (0.5 mg/kg AD05169) 0.643 0.172 0.613 0.161 N/A N/A N/A N/AGroup 14 (0.5 mg/kg AD05239) 0.438 0.065 0.542 0.014 N/A N/A N/A N/AGroup 15 (0.5 mg/kg AD05251) 0.125 0.013 0.132 0.037 0.157 0.033 0.1880.049 Group 16 (0.5 mg/kg AD05220) 0.211 0.012 0.201 0.087 0.230 0.0450.342 0.166

TABLE 35 Average Triglycerides Normalized to Pre-Dose from Example 11.Day 8 Day 15 Day 22 Day 29 Avg Std Dev Avg Std Dev Avg Std Dev Avg StdDev Group ID TG (+/−) TG (+/−) TG (+/−) TG (+/−) Group 1 (D5W) 0.9390.217 0.835 0.235 0.965 0.215 1.051 0.136 Group 2 (0.5 mg/kg AD05260)0.259 0.085 0.324 0.124 N/A N/A N/A N/A Group 3 (0.5 mg/kg AD05221)0.352 0.134 0.481 0.077 N/A N/A N/A N/A Group 4 (0.5 mg/kg AD05223)0.133 0.034 0.228 0.057 0.327 0.060 0.451 0.105 Group 5 (0.5 mg/kgAD05299) 0.352 0.134 0.481 0.086 N/A N/A N/A N/A Group 6 (0.5 mg/kgAD05283) 0.130 0.022 0.150 0.026 0.245 0.056 0.286 0.023 Group 7 (0.5mg/kg AD05284) 0.203 0.162 0.275 0.231 0.350 0.199 0.477 0.260 Group 8(0.5 mg/kg AD05167) 0.318 0.126 0.483 0.330 N/A N/A N/A N/A Group 9 (0.5mg/kg AD05168) 0.188 0.014 0.330 0.010 N/A N/A N/A N/A Group 10 (0.5mg/kg AD05171) 0.183 0.092 0.282 0.150 0.423 0.124 0.549 0.138 Group 11(0.5 mg/kg AD05258) 0.479 0.167 0.622 0.187 N/A N/A N/A N/A Group 12(0.5 mg/kg AD05259) 0.294 0.015 0.360 0.190 N/A N/A N/A N/A Group 13(0.5 mg/kg AD05169) 0.728 0.253 0.561 0.163 N/A N/A N/A N/A Group 14(0.5 mg/kg AD05239) 0.381 0.038 0.422 0.057 N/A N/A N/A N/A Group 15(0.5 mg/kg AD05251) 0.110 0.032 0.092 0.019 0.134 0.051 0.186 0.072Group 16 (0.5 mg/kg AD05220) 0.161 0.045 0.216 0.029 0.184 0.075 0.3580.141

TABLE 36 Average Total Cholesterol Normalized to Pre-Dose from Example11. Day 8 Day 15 Day 22 Day 29 Avg Total Std Dev Avg Total Std Dev AvgTotal Std Dev Avg Total Std Dev Group ID Chol (+/−) Chol (+/−) Chol(+/−) Chol (+/−) Group 1 (D5W) 0.769 0.127 0.684 0.182 0.835 0.167 0.7960.180 Group 2 (0.5 mg/kg AD05260) 0.320 0.081 0.367 0.072 N/A N/A N/AN/A Group 3 (0.5 mg/kg AD05221) 0.397 0.078 0.456 0.050 N/A N/A N/A N/AGroup 4 (0.5 mg/kg AD05223) 0.393 0.176 0.450 0.189 0.476 0.186 0.6060.193 Group 5 (0.5 mg/kg AD05299) 0.522 0.092 0.611 0.031 N/A N/A N/AN/A Group 6 (0.5 mg/kg AD05283) 0.413 0.058 0.372 0.053 0.450 0.1000.501 0.040 Group 7 (0.5 mg/kg AD05284) 0.430 0.270 0.444 0.241 0.5190.252 0.604 0.315 Group 8 (0.5 mg/kg AD05167) 0.464 0.231 0.557 0.382N/A N/A N/A N/A Group 9 (0.5 mg/kg AD05168) 0.298 0.034 0.388 0.012 N/AN/A N/A N/A Group 10 (0.5 mg/kg AD05171) 0.360 0.179 0.391 0.180 0.4730.147 0.538 0.141 Group 11 (0.5 mg/kg AD05258) 0.619 0.094 0.668 0.135N/A N/A N/A N/A Group 12 (0.5 mg/kg AD05259) 0.643 0.053 0.511 0.187 N/AN/A N/A N/A Group 13 (0.5 mg/kg AD05169) 0.731 0.089 0.636 0.013 N/A N/AN/A N/A Group 14 (0.5 mg/kg AD05239) 0.571 0.106 0.561 0.085 N/A N/A N/AN/A Group 15 (0.5 mg/kg AD05251) 0.248 0.065 0.287 0.147 0.260 0.0740.305 0.114 Group 16 (0.5 mg/kg AD05220) 0.400 0.081 0.438 0.048 0.4220.065 0.524 0.080

TABLE 37 Average HDL Normalized to Pre-Dose from Example 11. Day 8 Day15 Day 22 Day 29 Avg Std Dev Avg Std Dev Avg Std Dev Avg Std Dev GroupID HDL (+/−) HDL (+/−) HDL (+/−) HDL (+/−) Group 1 (D5W) 0.825 0.1190.893 0.217 0.912 0.179 0.886 0.262 Group 2 (0.5 mg/kg AD05260) 1.3560.337 1.331 0.435 N/A N/A N/A N/A Group 3 (0.5 mg/kg AD05221) 1.4830.266 0.953 0.166 N/A N/A N/A N/A Group 4 (0.5 mg/kg AD05223) 1.0580.198 1.032 0.300 0.856 0.209 0.868 0.349 Group 5 (0.5 mg/kg AD05299)1.456 0.345 1.137 0.460 N/A N/A N/A N/A Group 6 (0.5 mg/kg AD05283)2.494 0.174 2.150 0.465 1.731 0.397 1.738 0.156 Group 7 (0.5 mg/kgAD05284) 1.559 0.237 1.791 0.849 1.598 0.448 1.605 0.131 Group 8 (0.5mg/kg AD05167) 1.239 0.287 1.310 0.108 N/A N/A N/A N/A Group 9 (0.5mg/kg AD05168) 1.666 0.551 1.425 0.251 N/A N/A N/A N/A Group 10 (0.5mg/kg AD05171) 1.514 0.286 1.435 0.248 0.941 0.005 0.827 0.111 Group 11(0.5 mg/kg AD05258) 1.170 0.082 1.081 0.212 N/A N/A N/A N/A Group 12(0.5 mg/kg AD05259) 1.964 0.955 1.221 0.228 N/A N/A N/A N/A Group 13(0.5 mg/kg AD05169) 1.059 0.236 1.101 0.230 N/A N/A N/A N/A Group 14(0.5 mg/kg AD05239) 1.323 0.088 1.120 0.224 N/A N/A N/A N/A Group 15(0.5 mg/kg AD05251) 1.728 0.173 2.143 0.688 1.632 0.312 1.737 0.452Group 16 (0.5 mg/kg AD05220) 1.660 0.391 1.797 0.384 1.803 0.637 1.4790.333

TABLE 38 Average LDL Normalized to Pre-Dose from Example 11. Day 8 Day15 Day 22 Day 29 Avg Std Dev Avg Std Dev Avg Std Dev Avg Std Dev GroupID LDL (+/−) LDL (+/−) LDL (+/−) LDL (+/−) Group 1 (D5W) 0.699 0.1290.621 0.201 0.778 0.259 0.646 0.216 Group 2 (0.5 mg/kg AD05260) 0.3980.108 0.317 0.046 N/A N/A N/A N/A Group 3 (0.5 mg/kg AD05221) 0.4410.024 0.422 0.013 N/A N/A N/A N/A Group 4 (0.5 mg/kg AD05223) 0.4410.280 0.437 0.219 0.514 0.264 0.589 0.219 Group 5 (0.5 mg/kg AD05299)0.504 0.160 0.577 0.100 N/A N/A N/A N/A Group 6 (0.5 mg/kg AD05283)0.464 0.122 0.428 0.173 0.551 0.277 0.595 0.195 Group 7 (0.5 mg/kgAD05284) 0.394 0.258 0.404 0.179 0.398 0.214 0.471 0.290 Group 8 (0.5mg/kg AD05167) 0.572 0.306 0.678 0.536 N/A N/A N/A N/A Group 9 (0.5mg/kg AD05168) 0.329 0.067 0.374 0.017 N/A N/A N/A N/A Group 10 (0.5mg/kg AD05171) 0.303 0.186 0.280 0.134 0.401 0.113 0.429 0.180 Group 11(0.5 mg/kg AD05258) 0.669 0.105 0.702 0.140 N/A N/A N/A N/A Group 12(0.5 mg/kg AD05259) 0.588 0.208 0.407 0.211 N/A N/A N/A N/A Group 13(0.5 mg/kg AD05169) 0.626 0.116 0.672 0.057 N/A N/A N/A N/A Group 14(0.5 mg/kg AD05239) 0.473 0.138 0.488 0.124 N/A N/A N/A N/A Group 15(0.5 mg/kg AD05251) 0.254 0.147 0.344 0.257 0.234 0.063 0.306 0.166Group 16 (0.5 mg/kg AD05220) 0.364 0.043 0.439 0.045 0.461 0.157 0.4550.101

Example 12. In Vivo Testing of APOC3 RNAi Agents in Cynomolgus Monkeys

APOC3 RNAi agents were evaluated in cynomolgus monkeys. On day 1,cynomolgus macaque (Macaca fascicularis) primates (also referred toherein as “cynos”) were administered a single subcutaneous injection of0.3 mL/kg (approximately 2-3 mL volume, depending on animal mass)containing 3.0 mg/kg of APOC3 RNAi agent AD05876, formulated in saline.APOC3 RNAi agent AD05876 included modified nucleotides and a tridentateN-acetyl-galactosamine targeting ligand ((NAG37)s) conjugated to the5′-terminal end of the sense strand, as shown in Tables 4, 5, 6, and 7.

Two (2) cynos were tested (n=2). On days −8 (pre-dose), 29, and 50,liver biopsies were taken. For one of the monkeys, additional liverbiopsy samples were taken on day 15. On the date of each biopsycollection, cynos were anesthetized and ultrasound-guided liver biopsieswere performed to extract two or three liver tissue samplesapproximately 1 mm×4 mm in size. The biopsy samples were thenhomogenized, and levels of APOC3 mRNA in the cyno livers were measuredby RT-qPCR. Resulting values were then normalized to the pre-dose (inthis case, at day −8) APOC3 mRNA measurements. The resulting mRNA datais reflected in the following Tables 39 and 40:

TABLE 39 APOC3 mRNA Levels Normalized to Pre-Dose from Example 12 ofCyno #1 (cy0713). Relative APOC3 Low High mRNA Expression Error ErrorDay 29 0.125 0.003 0.003 Day 50 0.167 0.002 0.002

TABLE 40 APOC3 mRNA Levels Normalized to Pre-Dose from Example 12 ofCyno #2 (cy0716). Relative APOC3 Low High mRNA Expression Error ErrorDay 15 0.250 0.007 0.007 Day 29 0.112 0.005 0.00  Day 50 0.239 0.0030.003

Both of the cynos dosed with AD05876 showed a significant reduction inliver-specific APOC3 mRNA compared to pre-treatment measurements at allmeasured time points. On day 29, for example, the first cyno had areduction of APOC3 mRNA of approximately 87.5% (0.125), while the secondcyno had a reduction of approximately 88.8% (0.112), compared topre-dose levels.

Example 13. In Vivo Testing of APOC3 RNAi Agents in High Fructose CornSyrup (HFCS) Diet-Fed Rhesus Monkeys

APOC3 RNAi agent AD05876 was further evaluated in high-fructose cornsyrup (HFCS) diet-fed Rhesus monkeys. Rhesus monkeys were placed on anHFCS diet 37 days prior to dosing. These animals were known to developincreased plasma triglycerides greater than 180 mg/dL on the HFCS diet.On day 1 and again on day 29, four (4) Rhesus monkeys were administereda subcutaneous injection containing 4.0 mg/kg of APOC3 RNAi agentAD05876 formulated in saline (n=4). Two additional Rhesus monkeys wereadministered normal saline control. APOC3 RNAi agent AD05876 containedmodified nucleotides and included N-acetyl-galactosamine targetingligands conjugated to the 5′-terminal end of the sense strand, as shownin Tables 4, 5, 6, and 7.

Both fed and fasting blood samples were drawn for analysis, and fastingserum samples were analyzed on days −8 (predose), 8, and 15. Monkeyswere fasted overnight prior to each collection. APOC3 protein levels inserum were measured by ELISA assay (R&D Systems), according to themanufacturer's recommendations. Triglycerides, total cholesterol,high-density lipoprotein (HDL), and low-density lipoprotein (LDL) inserum were measured on a CobasR Integra 400 (Roche Diagnostics),according to the manufacturer's recommendations.

The APOC3 protein levels, triglyceride levels, total cholesterol levels,HDL levels, and LDL levels for each animal were normalized. Fornormalization, the level of APCO3 protein, triglyceride, HDL, and totalcholesterol, respectively, for each animal at a time point, was dividedby the pre-treatment level of expression in that animal (in this case atday −8) to determine the ratio of expression “normalized topre-treatment.”

Data from the study set forth in this Example are shown in the followingTables 41-45:

TABLE 41 Average APOC3 Protein Normalized to Pre-Treatment from Example13 (Fasted) Day 8 Day 15 Day 21 Day 29 Day 36 Avg Std Dev Avg Std DevAvg Std Dev Avg Std Dev Avg Std Dev Group ID APOC3 (+/−) APOC3 (+/−)APOC3 (+/−) APOC3 (+/−) APOC3 (+/−) Group 1 (saline control) 0.921 0.0070.902 0.009 0.922 0.026 0.905 0.025 0.922 0.006 Group 2 (4.0 mg/kgAD05876) 0.509 0.150 0.388 0.159 0.347 0.114 0.358 0.086 0.335 0.100

TABLE 42 Average TG Normalized to Pre-Treatment from Example 13 (Fasted)Day 8 Day 15 Day 21 Day 29 Day 36 Avg Std Dev Avg Std Dev Avg Std DevAvg Std Dev Avg Std Dev Group ID TG (+/−) TG (+/−) TG (+/−) TG (+/−) TG(+/−) Group 1 (saline control) 0.743 0.055 0.717 0.054 1.017 0.155 0.7580.263 0.659 0.111 Group 2 (4.0 mg/kg AD05876) 0.599 0.338 0.433 0.2860.395 0.247 0.435 0.212 0.408 0.269

TABLE 43 Average Total Cholesterol Normalized to Pre-Treatment fromExample 13 (Fasted) Day 8 Day 15 Day 21 Day 29 Day 36 Avg Total Std DevAvg Total Std Dev Avg Total Std Dev Avg Total Std Dev Avg Total Std DevGroup ID Chol (+/−) Chol (+/−) Chol (+/−) Chol (+/−) Chol (+/−) Group 1(saline control) 0.972 0.050 0.944 0.079 0.957 0.0.18 0.882 0.021 0.8940.038 Group 2 (4.0 mg/kg AD05876) 0.860 0.177 0.826 0.1119 0.825 0.0840.780 0.162 0.751 0.203

TABLE 44 Average HDL Normalized to Pre-Treatment from Example 13(Fasted) Day 8 Day 15 Day 21 Day 29 Day 36 Avg Total Std Dev Avg TotalStd Dev Avg Total Std Dev Avg Total Std Dev Avg Total Std Dev Group IDChol (+/−) Chol (+/−) Chol (+/−) Chol (+/−) Chol (+/−) Group 1 (salinecontrol) 1.082 0.098 1.071 0.111 1.003 0.158 1.025 0.131 1.027 0.071Group 2 (4.0 mg/kg AD05876) 1.370 0.267 1.445 0.479 1.465 0.537 1.3160.294 1.370 0.425

TABLE 45 Average LDL Normalized to Pre-Treatment from Example 13(Fasted) Day 8 Day 15 Day 21 Day 29 Day 36 Avg Total Std Dev Avg TotalStd Dev Avg Total Std Dev Avg Total Std Dev Avg Total Std Dev Group IDChol (+/−) Chol (+/−) Chol (+/−) Chol (+/−) Chol (+/−) Group 1 (salinecontrol) 0.892 0.060 0.928 0.046 0.823 0.034 0.804 0.076 0.804 0.172Group 2 (4.0 mg/kg AD05876) 0.777 0.129 0.856 0.136 0.842 0.186 0.7550.144 0.716 0.228

The Rhesus monkeys dosed with AD05876 at 4.0 mg/kg dosage levels showeda reduction in APOC3 protein compared to pre-treatment measurementsacross each of the measured time points. Further, substantial reductionsin both triglyceride levels and total cholesterol levels are also shown.For example, in one animal, triglycerides were reduced by approximately89% on day 22, and as shown in Table 42 above, mean triglyceride levelswere reduced by approximately 60% (0.395) on day 22. Additionally, meanHDL levels increased by approximately 47% on day 22 (see Table 44(1.465)), with one animal having a 2.2-fold increase in HDL levels.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

The invention claimed is:
 1. An RNAi agent for inhibiting expression ofan APOC3 gene, comprising: (a) an antisense strand comprising thenucleotide sequence selected from the group consisting of (5′→3′):(SEQ ID NO: 3) (i) UCACUGAGAAUACUGUCCCUC; and (SEQ ID NO: 5) (ii)UCACUGAGAAUACUGUCCCGU; and

(b) a sense strand comprising a nucleotide sequence that is at leastpartially complementary to the antisense strand; wherein all orsubstantially all of the nucleotides on the antisense strand, the sensestrand, or both the antisense strand and the sense strand are modifiednucleotides, and wherein the sense strand is linked to a targeting groupthat comprises N-acetyl-galactosamine.
 2. The RNAi agent of claim 1,wherein the modified nucleotides are 2′-O methyl nucleotides, 2′-fluoronucleotides, or combinations thereof.
 3. The RNAi agent of claim 1,wherein the targeting group comprises a structure selected from thegroup consisting of: (NAG13), (NAG13)s, (NAG18), (NAG18)s, (NAG24),(NAG24)s, (NAG25), (NAG25)s, (NAG26), (NAG26)s, (NAG27), (NAG27)s,(NAG28), (NAG28)s, (NAG29), (NAG29)s, (NAG30), (NAG30)s, (NAG31),(NAG31)s, (NAG32), (NAG32)s, (NAG33), (NAG33)s, (NAG34), (NAG34)s,(NAG35), (NAG35)s, (NAG36), (NAG36)s, (NAG37), (NAG37)s, (NAG38),(NAG38)s, (NAG39), (NAG39)s.
 4. The RNAi agent of claim 3, wherein thetargeting group comprises the structure of:


5. The RNAi agent of claim 3, wherein the targeting group is conjugatedto the sense strand.
 6. The RNAi agent of claim 5, wherein the targetinggroup is conjugated to the 5′ terminal end of the sense strand.
 7. TheRNAi agent of claim 1, wherein the sense strand is between 21 and 30nucleotides in length, and the antisense strand is between 21 and 30nucleotides in length.
 8. The RNAi agent of claim 7, wherein the sensestrand and the antisense strand are each between 21 and 27 nucleotidesin length.
 9. The RNAi agent of claim 8, wherein the sense strand andthe antisense strand are each between 21 and 24 nucleotides in length.10. The RNAi agent of claim 9, wherein the sense strand and theantisense strand are each 21 nucleotides in length.
 11. The RNAi agentof claim 10, wherein the RNAi agent has two blunt ends.
 12. The RNAiagent of claim 1, wherein the sense strand comprises one or two terminalcaps.
 13. The RNAi agent of claim 1, wherein the sense strand comprisesone or two inverted abasic residues.
 14. The RNAi agent of claim 1,wherein the sense strand comprises the nucleotide sequence selected fromthe group consisting of (5′→3′): SEQ ID NO: 16) (GAGGGACAGUAUUCUCAGUIA;(SEQ ID NO: 18) (ACGGGACAGUAUUCUCAGUIA; and (SEQ ID NO: 21)(GAGGGACAGUAUUCUCAGUGA;

wherein I represents an inosine nucleotide.
 15. The RNAi agent of claim14, wherein the sense strand further includes inverted abasic residuesat the 3′ terminal end and at the 5′ end of the nucleotide sequence. 16.The RNAi agent of claim 1, wherein the antisense strand comprises thenucleotide sequences selected from the group consisting of (5′→3′):(SEQ ID NO: 2) usCfsasCfuGfagaauAfcUfgUfcCfcUfsc; (SEQ ID NO: 4)usCfsasCfuGfagaauAfcUfgUfcCfcGfsu; and (SEQ ID NO: 6)usCfsascugagaauAfcUfgUfcCfcUfsc;

wherein a, c, g, and u represent 2′-O-methyl adenosine, cytidine,guanosine, or uridine, respectively; Af, Cf, Gf, and Uf represent2′-fluoro adenosine, cytidine, guanosine, or uridine, respectively; andsrepresents a phosphorothioate linkage.
 17. The RNAi agent of claim 16,wherein the sense strand comprises the nucleotide sequence selected fromthe group consisting of (5′→3′): (SEQ ID NO: 15)gagggacaGfUfAfuucucaguia; (SEQ ID NO: 17) acgggacaGfUfAfuucucaguia;(SEQ ID NO: 19) gagggacaGfuAfuUfcucaguia; and (SEQ ID NO: 20)gagggacaGfUfAfuucucaguga;

wherein a, c, g, i, and u represent 2′-O-methyl adenosine, cytidine,guanosine, inosine, or uridine, respectively; Af, Cf, Gf, If, and Ufrepresent 2′-fluoro adenosine, cytidine, guanosine, inosine or uridine,respectively; and s represents a phosphorothioate linkage.
 18. The RNAiagent of claim 17, wherein the sense strand further includes an invertedabasic residue at the 3′ terminal end and/or at the 5′ end of thenucleotide sequence.
 19. The RNAi agent of claim 1, wherein the RNAiagent has the duplex structure selected from the group consisting of:AD05251 (SEQ ID NOs: 2 and 501); AD05876 (SEQ ID NOs: 4 and 572);AD05769 (SEQ ID NOs: 6 and 557); and AD05169 (SEQ ID NOs: 2 and 482).20. The RNAi agent of claim 19, wherein the RNAi agent has the duplexstructure of AD05251 (SEQ ID NOs: 2 and 501).
 21. A compositioncomprising the RNAi agent of claim 1, wherein the composition comprisesa pharmaceutically acceptable excipient.
 22. A composition comprisingthe RNAi agent of claim 16, wherein the composition comprises apharmaceutically acceptable excipient.
 23. A composition comprising theRNAi agent of claim 17, wherein the composition comprises apharmaceutically acceptable excipient.
 24. The composition of claim 21,wherein the composition further comprises a second RNAi agent forinhibiting the expression of an APOC3 gene.
 25. The composition of claim21, wherein the composition further comprises one or more additionaltherapeutics.
 26. A method for inhibiting expression of an APOC3 gene ina cell, the method comprising introducing into a cell an effectiveamount of the composition of claim
 21. 27. The method of claim 26,wherein the cell is within a subject.
 28. The method of claim 27,wherein the subject is a human subject.
 29. A method of treating anAPOC3-related disease or disorder, the method comprising administeringto a human subject in need thereof a therapeutically effective amount ofthe composition of claim
 21. 30. The method of claim 29, wherein thedisease is a cardiometabolic disease.
 31. The method of claim 30,wherein the cardiometabolic disease is hypertriglyceridemia, obesity,hyperlipidemia, abnormal lipid and/or abnormal cholesterol metabolism,atherosclerosis, cardiovascular disease, coronary artery disease,hypertriglyceridemia induced pancreatitis, metabolic syndrome, type IIdiabetes mellitus, familial chylomicronernia syndrome, or familialpartial lipodystrophy.
 32. The method of claim 29, wherein the RNAiagent is administered at a dose of about 0.05 mg/kg to about 5.0 mg/kgof body weight of the human subject.
 33. The method of claim 29, whereinthe RNAi agent is administered in two or more doses.
 34. The method ofclaim 29, wherein the dose is administered by subcutaneous injection.35. A method of lowering triglyceride levels in a subject, the methodcomprising administering to the subject an effective amount of acomposition of claim
 21. 36. A method of lowering cholesterol levels ina subject, the method comprising administering to the subject aneffective amount of a composition of claim
 21. 37. A method of loweringlow density lipoprotein (LDL) levels in a subject, the method comprisingadministering to the subject an effective amount of a composition ofclaim
 21. 38. The RNAi agent of claim 16, wherein the antisense strandcomprises the nucleotide sequence (5′→3′)usCfsasCfuGfagaauAfcUfgUfcCfcUfsc (SEQ ID NO:2); wherein a, c, g, and urepresent 2′-O-methyl adenosine, cytidine, guanosine, or uridine,respectively; Af, Cf, Gf, and Uf represent 2′-fluoro adenosine,cytidine, guanosine, or uridine, respectively; and s represents aphosphorothioate linkage.
 39. The RNAi agent of claim 38, wherein thesense strand comprises the nucleotide sequence (5′→3′)gagggacaGfUfAfuucucaguia (SEQ ID NO:15), wherein a, c, g, i, and urepresent 2′-O-methyl adenosine, cytidine, guanosine, inosine, oruridine, respectively; Af, Cf, Gf, and Uf represent 2′-fluoro adenosine,cytidine, guanosine, or uridine, respectively; and wherein the sensestrand optionally includes inverted abasic residues at the 3′ terminalend and/or at the 5′ end of the nucleotide sequence.
 40. The RNAi agentof claim 38, wherein the sense strand comprises the nucleotide sequence(5′→3′) (NAG37)s(invAb)sgagggacaGfUfAfuucucaguias(invAb) (SEQ IDNO:501), wherein a, c, g, i, and u are 2′-O-methyl adenosine, cytidine,guanosine, inosine, or uridine, respectively; Af, Cf, Gf, and Uf are2′-fluoro adenosine, cytidine, guanosine, or uridine, respectively; s isa phosphorothioate linkage; (invAb) is an inverted abasic deoxyriboseresidue, and (NAG37)s has the following chemical structure:


41. The RNAi agent of claim 38, wherein the sense strand comprises thenucleotide sequence (5′→3′) gagggacaGfUfAfuucucaguga (SEQ ID NO:20),wherein a, c, g, and u represent 2′-O-methyl adenosine, cytidine,guanosine, or uridine, respectively; Af, Cf, Gf, and Uf represent2′-fluoro adenosine, cytidine, guanosine, or uridine, respectively; andwherein the sense strand optionally includes inverted abasic residues atthe 3′ terminal end and/or at the 5′ end of the nucleotide sequence. 42.The RNAi agent of claim 38, wherein the sense strand comprises thenucleotide sequence (5′→3′)(NAG37)s(invAb)sgagggacaGfUfAfuucucagugas(invAb) (SEQ ID NO:482),wherein a, c, g, i, and u are 2′-O-methyl adenosine, cytidine,guanosine, inosine, or uridine, respectively; Af, Cf, Gf, and Uf are2′-fluoro adenosine, cytidine, guanosine, or uridine, respectively; s isa phosphorothioate linkage; (invAb) is an inverted abasic deoxyriboseresidue, and (NAG37)s has the following chemical structure:


43. The RNAi agent of claim 16, wherein the antisense strand comprisesthe nucleotide sequence (5′→3′) usCfsasCfuGfagaauAfcUfgUfcCfcGfsu (SEQID NO:4); wherein a, c, g, and u represent 2′-O-methyl adenosine,cytidine, guanosine, or uridine, respectively; Af, Cf, Gf, and Ufrepresent 2′-fluoro adenosine, cytidine, guanosine, or uridine,respectively; and s represents a phosphorothioate linkage.
 44. The RNAiagent of claim 43, wherein the sense strand comprises the nucleotidesequence (5′→3′) acgggacaGfUfAfuucucaguia (SEQ ID NO:17), wherein a, c,g, i, and u represent 2′-O-methyl adenosine, cytidine, guanosine,inosine, or uridine, respectively; Af, Cf, Gf, and Uf represent2′-fluoro adenosine, cytidine, guanosine, or uridine, respectively; andwherein the sense strand optionally includes inverted abasic residues atthe 3′ terminal end and/or at the 5′ end of the nucleotide sequence. 45.The RNAi agent of claim 43, wherein the sense strand comprises thenucleotide sequence (5′→3′)(NAG37)s(invAb)sacgggacaGfUfAfuucucaguias(invAb) (SEQ ID NO:572),wherein a, c, g, i, and u are 2′-O-methyl adenosine, cytidine,guanosine, inosine, or uridine, respectively; Af, Cf, Gf, and Uf are2′-fluoro adenosine, cytidine, guanosine, or uridine, respectively; s isa phosphorothioate linkage; (invAb) is an inverted abasic deoxyriboseresidue, and (NAG37)s has the following chemical structure:


46. The RNAi agent of claim 16, wherein the antisense strand comprisesthe nucleotide sequence (5′→3′) usCfsascugagaauAfcUfgUfcCfcUfsc (SEQ IDNO:6); wherein a, c, g, and u represent 2′-O-methyl adenosine, cytidine,guanosine, or uridine, respectively; Af, Cf, Gf, and Uf represent2′-fluoro adenosine, cytidine, guanosine, or uridine, respectively; ands represents a phosphorothioate linkage.
 47. The RNAi agent of claim 46,wherein the sense strand comprises the nucleotide sequence (5′→3′)gagggacaGfuAfuUfcucaguia (SEQ ID NO:19), wherein a, c, g, i, and urepresent 2′-O-methyl adenosine, cytidine, guanosine, inosine, oruridine, respectively; Af, Cf, Gf, and Uf represent 2′-fluoro adenosine,cytidine, guanosine, or uridine, respectively; and wherein the sensestrand optionally includes inverted abasic residues at the 3′ terminalend and/or at the 5′ end of the nucleotide sequence.
 48. The RNAi agentof claim 46, wherein the sense strand comprises the nucleotide sequence(5′→3′) (NAG37)s(invAb)sgagggacaGfuAfuUfcucaguias(invAb) (SEQ IDNO:557), wherein a, c, g, i, and u are 2′-O-methyl adenosine, cytidine,guanosine, inosine, or uridine, respectively; Af, Cf, Gf, and Uf are2′-fluoro adenosine, cytidine, guanosine, or uridine, respectively; s isa phosphorothioate linkage; (invAb) is an inverted abasic deoxyriboseresidue, and (NAG37)s has the following chemical structure:


49. The RNAi agent of claim 19, wherein the RNAi agent has the duplexstructure of AD05169 (SEQ ID NOs: 2 and 482).
 50. The RNAi agent ofclaim 19, wherein the RNAi agent has the duplex structure of AD05876(SEQ ID NOs: 4 and 572).
 51. The RNAi agent of claim 19, wherein theRNAi agent has the duplex structure of AD05769 (SEQ ID NOs: 6 and 557).